Methods to produce bunyavirus replicon particles

ABSTRACT

The invention relates to methods of producing infectious bunyavirus replicon particles. These bunyavirus replicon particles are safe and can be used outside a biosafety containment. The invention further relates to recombinant bunyavirus replicon particles and uses of these recombinant bunyavirus replicon particles.

RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/NL2011/050631, filed Sep. 20, 2011, published in English, andclaims the benefit of European Application Number 10177709.2, filed onSep. 20, 2010 and U.S. Application No. 61/468,597, filed on Mar. 29,2011, the entire teachings of the above applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 18, 2014, isnamed P92482US10seqlist_ST25 and is 75,177 bytes in size.

FIELD

The invention relates to the field of recombinant viruses. Morespecifically, the invention relates to methods for generatingrecombinant bunyavirus particles that are incapable of autonomousspread. The resulting bunyavirus particles can be used as a vaccine toprotect a mammal against infectious disease mediated by bunyavirus, andcan be used as vector for the transduction of a foreign gene.

The Bunyaviridae family is divided into five genera, of which four(Orthobunyavirus, Nairovirus, Phlebovirus and Hantavirus) includenumerous virus species that are capable of causing severe disease inboth animals and humans. Well known examples are hantaanvirus (HTNV,Hantavirus genus), Crimean-Congo hemorrhagic fever virus (CCHFV,Nairovirus genus) and Rift Valley fever virus (RVFV, Phlebovirus genus).

In the veterinary field, RVFV is one of the most feared bunyaviruses.Transmission of RVFV between ruminants occurs via the bite of infectedmosquitoes, whereas infection of humans is believed to occurpredominantly via aerosols released from contaminated animal products.Mortality rates in adult ruminants vary from 10 to 20%. Mortality ratesin unborn and young animals can be more dramatic, approaching 100%.Although the human mortality rate is historically estimated at about 2%,considerably higher mortality rates were reported after recentoutbreaks. Although the virus is currently confined to the Africancontinent and the Arabian Peninsula, mosquitoes that transmit RVFV arenot restricted to these areas. This explains the growing concern forRVFV incursions into other parts of the world, including Europe, Asiaand the Americas.

Bunyavirus family members contain a three-segmented RNA genome, which iscomprised of a large (L), a medium (M) and a small (S) segment. Allfamily members produce the structural nucleocapsid (N) protein from theS genome segment, the viral polymerase protein from the L genome segmentand the G1 and G2 structural glycoproteins from the M genome segment.Nonstructural proteins are encoded by the S (referred to as NSs) and Msegments (referred to as NSm) of phleboviruses and orthobunyaviruses.Interestingly, the M segment of nairoviruses encodes several structuralas well as non-structural proteins and glycoprotein synthesis andprocessing of these viruses is quite distinct from other members of theBunyaviridae family. However, the skilled person is without any doubtsable to apply the teachings of the present invention for the generationof nairoviruses replicon particles.

The RVFV M segment encodes the structural glycoproteins G2 (generallyreferred to as Gn) and G1 (generally referred to as Gc) and at least twonon-structural proteins, which are collectively referred to as NSm. TheM segment encodes a singly mRNA with multiple translation initiationsites. The translation products are Gn, Gc and the at least two NSmproteins. The viral genomic segments contain untranslated regions (UTRs)on both the 3′ and the 5′ ends that serve as promoters for replicationof the segment and for transcription of the encoded reading frames.

The recent establishment of a reverse-genetics system for RVFV hasprovided important new insights into its biology. A few years after thefirst successful rescue of the complete RVFV from cloned cDNA, Habjan etal. described the packaging of a reporter minigenome into virus-likeparticles (VLPs) (Habjan et al. 2009. Virology 385, 400-408). The VLPswere produced by transient expression of the NSm, Gn, Gc, N and Lproteins in the presence of a reporter minigenome. The N and L proteinsinduced expression of a reporter protein from the replicated minigenomeand the structural glycoproteins subsequently packaged the minigenomeinto VLPs. These VLPs were shown to transport the reporter minigenome toreceiving cells. Whereas primary transcription in these cells wasobserved, replication of the minigenome was dependent on the productionof the N and L proteins from transfected plasmids. Cells that are onlyinfected with the VLPs and which are not cotransfected with constructsexpressing N and L proteins, show limited expression of viral protein.The viral genome is not replicated and there is no amplification of thegenome in these cells. Therefore, only primary transcription of theviral genome occurs in these cells.

Although RVFV, HTNV and CCHFV cause severe disease with high casefatality in humans, no vaccines are available for the prevention ofthese diseases in humans, and no antiviral agents are registered forpost-exposure treatment. The development of such control tools isseverely complicated by the fact that these viruses must be handledunder high biosafety containment. RVFV also causes severe disease withhigh case fatality in livestock. Effective, but not safe, vaccines areregistered for use outside the African continent.

Therefore, there is an urgent need to develop methods and means for safeand efficient production of bunyavirus particles.

The present invention discloses non-spreading bunyavirus repliconparticles that are capable of autonomous genome replication. Bunyavirusreplicon particles (BRPs) were produced by trans-complementation of thestructural glycoproteins G1 (G1 of RVFV is referred to as Gc) and G2 (G2of RVFV is referred to as Gn) in both the presence and absence of theNSm coding-regions. BRPs were produced up to titers of 10E7 infectiousparticles/ml. The resultant particles can be used to study all aspectsof the viral life cycle outside biosafety containment facilities both invitro and in vivo. Furthermore, the particles can be used invirus-neutralization tests that can be performed outside biosafetycontainment facilities and the antigens produced can be used for ELISAsand other serological tests. Moreover, the methods described here willfacilitate the development of therapeutics and vaccines that optimallycombine the safety of inactivated vaccines with the efficacy oflive-attenuated vaccines. The methods can also be applied for theestablishment of novel gene-delivery systems.

In a first aspect, the invention provides a method for generating arecombinant non-spreading BRP, the method comprising: A) providing aeukaryotic cell with growth medium; B1) providing the eukaryotic cellwith sufficient DNA-dependent RNA polymerase, for example T7 polymerase;B2) providing the eukaryotic cell with sufficient bunyavirus (NSm)GnGcprotein; B3) providing the eukaryotic cell with a vector that comprisesa copyDNA (cDNA) of a bunyavirus L genome segment which is flanked atthe 5′ end by a T7 promoter, and at the 3′ end with cDNA encoding aribozyme sequence; B4) providing the eukaryotic cell with a vector thatcomprises a cDNA of a Bunyavirus S genome segment or part of aBunyavirus S genome segment at least comprising the N gene and the 3′and 5′ UTRs, which are flanked at the 5′ end by a T7 promoter and at the3′ end with cDNA encoding a ribozyme sequence; and, optionally, B5)providing the eukaryotic cell with a vector that comprises a cDNA of aBunyavirus M genome segment from which the GnGc coding region has beenfunctionally inactivated, the cDNA encoding the genome segment inbetween the 3′ and 5′ UTRs is flanked at the 5′ end by a T7 promoter andat the 3′ end with cDNA encoding a ribozyme sequence; C) generating arecombinant Bunyavirus replicon particle which can be isolated from thegrowth medium; wherein the sequence of steps of B1, B2, B3, B4 and B5 israndom and all or part of these steps may be performed simultaneously.

The cDNAs of the bunyavirus L, S and M genome segments are present inthe vector in the genomic sense orientation or in the antigenomic senseorientation. When the cDNAs of the bunyavirus L, S and M genome segmentsare present in the vector in the genomic sense orientation, it ispreferred that the cells are provided with plasmids that produce the Nand L proteins.

The cDNAs of the bunyavirus genome segments in a method for generating arecombinant bunyavirus replicon particle according to the invention areflanked at the 5′ end by a promoter sequence for a DNA-dependent RNApolymerase. Said promoter sequence for a DNA-dependent RNA polymerase isselected from any known promoter sequences of DNA-dependent RNApolymerases such as, but not limited to, the promoter sequence of aeukaryotic RNA polymerase I such as, for example, a promoter for murineRNA polymerase I, SP6, T3 and T7. For example, a method according to theinvention in which a eukaryotic cell is provided with sufficient T3polymerase and in which Bunyavirus genomic segments are flanked by a T3promoter sequence or a method according to the invention in which aeukaryotic cell is provided with sufficient SP6 polymerase and in whichcDNA encoding bunyavirus genomic segments are flanked by a SP6 promotersequence, are also provided by the invention. T7 is a preferredDNA-dependent RNA polymerase. Although the description and the claimsrefer to T7 polymerase, it is to be understood that the invention is notlimited to T7 polymerase but includes other DNA-dependent RNA polymerasesuch as, for example, T3 polymerase and SP6 polymerase. A preferredpromoter sequence for a T7 polymerase is TAATACGACTCACTATAG.

Copy DNA of bunyavirus genomic segments or fragments thereof are flankedat the 3′ ends by a cDNA encoding a ribozyme sequence that mediates 3′end formation of the RNA by self-cleavage of the nascent RNA. Apreferred ribozyme sequence is a hepatitis delta virus (HDV) ribozymesequence. A termination sequence that mediates termination of theDNA-dependent RNA polymerase may further be present distal to the cDNAencoding the ribozyme sequence. In a preferred embodiment, theDNA-dependent RNA polymerase is T7 polymerase and the terminationsequence is a T7 transcription termination sequence. Promoter sequencesfor DNA-dependent RNA polymerases, such as T7 polymerase, andtermination sequences such as a T7 transcription termination sequence,are known to the skilled person.

The term “recombinant bunyavirus replicon particle” refers to abunyavirus particle that comprises at least a bunyavirus L-genomesegment and (at least) a part of a bunyavirus S genome segmentcomprising the N-gene and the 3′ and 5′ UTRs. These genomic segmentsencode the proteins that are required for transcription and replicationof these viral genomic segments in an infected cell, resulting inreplication and thus amplification of the L and S genome segments in aninfected cell. Cells that are infected with a recombinant bunyavirusreplicon particle according to the invention express high levels of atleast the bunyavirus L and N proteins.

The term “L-genome segment” refers to a substantially complete L-genomesegment. The term “substantially complete” is used to indicate that theL genome segment comprises cis-acting elements that mediate replicationof the L genome segment and that mediate functional expression of theL-gene. The term “substantially complete” indicates that sequences thatare not involved in replication of the L genome segment or in functionalexpression of the L-gene may be deleted or substituted. The term“functional expression” refers to expression of an L protein, a viralRNA-dependent RNA polymerase, that is able to mediate replication andtranscription of a bunyavirus genome segment or bunyavirus minigenome.The term “minigenome” refers to an RNA molecule that comprises the 5′and 3′ regions of a bunyavirus genome segment that function inreplication of the segment, but which lacks at least one bunyaviruscoding region that is present on the wildtype genome segment. Aminigenome may further comprise a foreign gene such as, but not limitedto, a marker gene such as a Fluorescent Protein, beta-glucuronidase andbeta-galactosidase. The term “L genome segment from which the L codingregion has been functionally inactivated” refers to an L genome segment,comprising the 3′ and 5′ UTRs of the L genome segment.

The term “S genome segment comprising the N gene” refers to an S genomesegment, comprising the untranslated regions of both the 3′ and the 5′end of the S genome segment and at least the nucleotide sequences forexpression of the N protein, such as nucleotide sequences fortranscription of the N-gene and translation of the N-gene-transcript.The term “S genome segment from which the NSs and N coding regions havebeen functionally inactivated” refers to an S genome segment, comprisingthe 3′ and 5′ UTRs and the untranslated intergenic region of the Sgenome segment.

The term “M genome segment from which the GnGc coding region has beenfunctionally inactivated” refers to an M genome segment, comprising theuntranslated regions of both the 3′ and the 5′ end of the M genomesegment.

Bunyavirus L-, S- and M-genome segments are preferably cloned instandard eukaryotic expression vectors, whereby the genome segments areflanked by a DNA-dependent RNA polymerase promoter, preferably a T7promoter, and a HDV ribozyme sequence. Suitable vectors comprisepBluescript (Stratagene), pUC plasmids such as preferably pUC57(Genscript), and medium and low-copy number vectors such as pBR322 andderivates thereof (Mobitec, Germany), pACYC184 (Chang and Cohen, 1978)and pCC1 (Epicentre Biotechnologies, Madison, Wis.). Some Bunyavirusgenome segments, especially L-genome segments, are more stable whencloned in medium or low copy number vectors preferably pCC1.

The term functionally inactivated refers to a gene of which the activityof the encoded RNA or protein is less than 10% under the same conditionsof the activity of the encoded RNA or protein of a gene that is notfunctionally inactivated, more preferred less than 5%, more preferredless than 2%, more preferred less than 1%. The term “functionallyinactivated” most preferably indicates a gene that is not expressedbecause it is not transcribed or not translated, or a gene of which theencoded protein is not active, for example by alteration or deletion ofone or more nucleotides within the coding region of the gene. The term“functionally inactivated gene” preferably is a gene of which part orall of the coding sequences have been deleted.

The order in which a eukaryotic cell is provided with sufficient T7polymerase, sufficient bunyavirus (NSm)GnGc protein, a bunyavirus Lgenome segment, a bunyavirus S genome segment comprising at least theN-gene and, optionally, a Bunyavirus M genome segment from which the(NSm)GnGc coding region has been functionally inactivated, is random.All or part of these steps may be performed subsequent to one another,or simultaneously. It will be clear to the skilled person that thebunyavirus L genome segment and/or a bunyavirus S genome segment maycomprise a functional deletion of the L-gene and/or N-gene,respectively, when the cell is provided with RNA polymerase enzyme thatis encoded by a Bunyavirus L-genome segment and/or a N protein that isencoded by a bunyavirus S-genome segment.

For practical reasons, it is preferred that a eukaryotic cell isprovided first with a bunyavirus L genome segment, a bunyavirus S genomesegment comprising the N-gene and, optionally, a bunyavirus M genomesegment from which the (NSm)GnGc coding region has been functionallyinactivated, The resulting cell line harbouring a Bunyavirus L genomesegment, a bunyavirus S genome segment comprising the N-gene and,optionally, a bunyavirus M genome segment from which the (NSm)GnGccoding region has been functionally inactivated, is subsequentlyprovided with sufficient bunyavirus (NSm)GnGc protein to mediateefficient packaging of the bunyavirus genome segments into BRPs.

Alternatively, a cell line is first provided with sufficient T7polymerase and sufficient bunyavirus (NSm)GnGc protein by infection ortransfection of a construct encoding these proteins, followed byprovision of the cell with a bunyavirus L genome segment, a bunyavirus Sgenome segment comprising the N-gene and, optionally, a bunyavirus Mgenome segment from which the (NSm)GnGc coding region has beenfunctionally inactivated.

The term “sufficient T7 polymerase” refers to the amount of T7polymerase that is provided to a eukaryotic cell that is sufficient tomediate efficient transcription of cDNA molecules encoding thebunyavirus L genome segment and the complete S genome segment or thepart of a bunyavirus S genome segment at least comprising the N gene,that are flanked by a T7 promoter sequence and cDNA encoding a HDVribozyme. It was found that rescue of a bunyavirus by providing aeukaryotic cell stably expressing T7 RNA polymerase under control of acytomegalovirus promoter such as, for example, the BSR T7/5 cell line(Buchholz et al. 1999. J. Virol. 73: 251-259) with one or more vectorsthat comprise a bunyavirus L genome segment, a bunyavirus M genome,and/or a bunyavirus S genome segment, or functional parts of one or moreof the bunyavirus genome segments, was inefficient.

An improved and reproducible rescuing efficiency was obtained wheneukaryotic cells were freshly infected or transfected with an expressionvector that encodes the T7 polymerase.

Therefore, in a preferred method according to the invention, theeukaryotic cell is provided with sufficient T7 polymerase by freshlytransfecting or infecting the eukaryotic cell with an expression vectorthat encodes the T7 polymerase.

In one embodiment, the expression vector is a plasmid that encodes theT7 polymerase. Suitable plasmids are, for example, pCAGGS, and pcDNA. Ina preferred embodiment, the expression vector is a recombinant virus orviral vector that encodes the T7 polymerase. A suitable virus or viralvector is, for example, a replication defective retroviral vector suchas a lentiviral vector, for example a HIV-based vector or an EIAV-basedvector, or a replication defective MMLV-based vector. A further suitablevirus or viral vector is provided by a replication defective adenoviralvector and a baculoviral vector. A preferred virus or viral vector is areplication defective poxvirus such as, for example, a vaccinia-basedvirus. In a most preferred method according to the invention, theeukaryotic cell is provided with sufficient T7 polymerase by infectingthe eukaryotic cell with a fowlpoxvirus (FPV)-based expression vectorthat encodes the T7 polymerase. The FPV may be replication competent orreplication defective.

Without being bound by theory, a reason for the improved andreproducible rescuing efficiency by using a FPV-based expression vectoris that the level of T7 polymerase is sufficiently high to allowefficient transcription of the bunyavirus cDNA genome segments. Afurther reason could be that FPVs produce their own capping enzyme.Capping of the T7 transcripts could stabilize the bunyaviral RNA that isproduced from the cDNA, protecting the RNA from degradation.

A further advantage of a FPV is that it belongs to the genus Avipoxvirusand is capable of spreading in avian cells. In non-avian cells such as,for example, mammalian cells, FPV replication is abortive with noevidence of production of infectious virus. Therefore, when theeukaryotic cell is a non-avian eukaryotic cell, a replication competentFPV-based expression vector or a replication deficient FPV-basedexpression vector is preferably used for a method of the invention. Whenthe eukaryotic cell is an avian eukaryotic cell, it is preferred that areplication deficient FPV-based expression vector is used for a methodof the invention.

The term “providing (NSm)GnGc protein” indicates that at least thebunyavirus Gn and Gc proteins are provided. In addition to the Gn and Gcprotein, also one or more NSm proteins may be provided.

The term “sufficient bunyavirus (NSm)GnGc protein” refers to the amountof (NSm)GnGc proteins that are provided to a eukaryotic cell that issufficient to mediate efficient packaging of the bunyavirus genomesegments in a recombinant bunyavirus replicon particle. A cell can beprovided with sufficient bunyavirus (NSm)GnGc proteins by transfectingor infecting the cell with a vector that mediates expression of thebunyavirus (NSm)GnGc proteins. If a Bunyavirus M genome segment fromwhich the (NSm)GnGc coding region has been functionally inactivated, ispresent in the eukaryotic cell, it is preferred that there is nosequence overlap between the bunyavirus (NSm)GnGc protein-encodingsequence in the eukaryotic cell and the bunyavirus M genome segment toprevent the generation of a packaging-competent bunyavirus.

A eukaryotic cell is preferably provided with sufficient bunyavirus(NSm)GnGc proteins by transfecting the eukaryotic cell with anexpression plasmid encoding the bunyavirus (NSm)GnGc proteins. Saidexpression plasmid preferably comprises a promoter region comprisingregulatory sequences that control the expression of the bunyavirus(NSm)GnGc proteins. Suitable promoter sequences are known in the art,including, but not limiting to, promoter sequences from a virus such ascytomegalovirus (CMV), or a promoter region from a housekeeping genesuch as beta-actin, for example a chicken actin promoter. Ifintroduction of such expression vector(s) results in death of cells thatproduce high levels of the proteins due to their toxicity, end-pointdilution yields clones that express tolerable levels of these proteins.It was found that the selected cells were tolerable for higherexpression levels provided by subsequent (re)introduction of(NSm)GnGc-producing expression vector(s).

The invention further provides a eukaryotic replicon cell lineexpressing (NSm)GnGc-proteins and comprising the bunyavirus L genomesegment and the bunyavirus S genome segment or at least part of abunyavirus S genome segment comprising the N-gene and the 3′ and 5′UTRs. It was found that low levels of (NSm)GnGc was sufficient toprevent loss of the bunyavirus genome segments from the cells. Withoutbeing bound by theory, expression of the (NSm)GnGc-proteins, albeit atlow levels, allowed production of BRPs which continuously re-infectedcells. To produce BRPs, the eukaryotic replicon cell line is providedwith sufficient bunyavirus (NSm)GnGc protein by repetitive introductionof a vector providing bunyavirus (NSm)GnGc protein.

In a more preferred embodiment, the eukaryotic cell in a methodaccording to the invention is provided with bunyavirus (NSm)GnGcproteins by infecting the eukaryotic cell with a viral vector thattransduces the bunyavirus (NSm)GnGc proteins. In one embodiment, saidviral vector is an adenovirus-based vector, a retrovirus-based vector ora herpesvirus-based vector. A preferred viral vector that transduces thebunyavirus (NSm)GnGc proteins is a paramyxovirus-based vector. Apreferred paramyxovirus is of the genus Avulavirus, which includes avianparamyxovirus. A preferred avian paramyxovirus is Newcastle diseasevirus (NDV). A preferred NDV comprises a recombinant cDNA clone of NDVstrain LaSota, named NDFL (Peeters et al. 1999. J. Virol. 73:5001-5009), in which a codon-optimized GnGc gene is flanked by newlyintroduced transcription start and stop sequences (Kortekaas et al.2010. Vaccine 28:4394-4401). A further preferred NDV is a vector derivedfrom a recombinant virulent strain such as, for example, GB Texas,Italien, Milano and Herts '33/56, that transduces (NSm)GnGc proteins.The vector preferably is a non-replicative or non-spreading NDV. Apreferred vector comprises a genome from a recombinant virulent NDVstrain with a deletion in the gene encoding the HN-protein. The viralvector is produced in a cell line that trans-complements the HN protein.

In a further preferred embodiment, the eukaryotic cell is provided withbunyavirus (NSm)GnGc proteins by infecting the eukaryotic cell with arecombinant viral vector that transduces the bunyavirus (NSm)GnGcproteins, followed by selection of a cell in which the recombinant viralvector is persistently present without causing overt cytopathogeniceffect. Proteins encoded by the virus such as, for example, bunyavirus(NSm)GnGc proteins, are expressed during the persistent infection. Itwas found that a persistently infected cell tolerates higher expressionlevels of bunyavirus GnGc proteins, compared to a cell that is stablytransformed with an expression vector expressing bunyavirus GnGcproteins.

A preferred viral vector for generating a persistently infected cell isbased on a herpesvirus, for example a herpes simplex virus, Epstein-Barvirus, or varicella zoster virus, on a retrovirus such as, for exampleHIV, EIAV, or MMLV, or on a paramyxovirus, for example an Avulavirus,which includes avian paramyxovirus. A preferred avian paramyxovirus isNewcastle disease virus (NDV). A preferred NDV comprises a recombinantcDNA clone of NDV strain LaSota, named NDFL (Peeters et al. 1999. J.Virol. 73: 5001-5009), in which a codon-optimized GnGc gene is presentflanked by newly introduced transcription start and stop sequences(Kortekaas et al. 2010. Vaccine 28:4394-4401).

A eukaryotic cell may be transiently or stably expressing bunyavirus(NSm)GnGc proteins. It was found initially that a eukaryotic cell thatwas stably transformed with an expression plasmid mediating highexpression levels of bunyavirus (NSm)GnGc proteins could not beobtained. This may in part be explained by our observation thatconstitutive high expression levels of GnGc might not be tolerated ineukaryotic cells. However, expression from a virus that persistentlyinfected these eukaryotic cells was tolerated.

To enable the generation of a stable cell line, a eukaryotic cell ispreferably provided with bunyavirus (NSm)GnGc proteins by transfectingor infecting the eukaryotic cell with an expression vector that providesconditional expression of the bunyavirus (NSm)GnGc proteins. The term“conditional expression” is known to the skilled person and refers to acontrolled expression of a protein, which is not, or only at low levelexpressed under a first condition, but of which the expression isincreased under a second condition.

In a preferred conditional expression system, expression of bunyavirus(NSm)GnGc proteins is dependent on the presence of an inducer or theabsence of an inhibitor. Several inducible gene expression systems arecurrently available that can be used to control expression of (NSm)GnGcproteins. Tet-On and Tet-Off expression systems (for example Tet-On® andTet-Off® Advanced Inducible Gene Expression Systems, Clontech) can beused for inducible expression of a gene of interest. In these systemsexpression of the transcriptional activator (tTA) is regulated by thepresence (Tet-On) or absence (Tet-Off) of tetracycline (TC) or aderivative of tetracycline such as doxycycline (dox). The tTA iscomposed of the Escherichia coli Tet repressor protein (TetR) and Herpessimplex virus transactivating domain VP16. tTA regulates transcriptionof a gene of interest under the control of a tetracycline-responsiveelement (TRE) consisting of the Tet operator (TetO) DNA sequence and apromoter sequence, for instance the human cytomegalovirus (hCMV)promoter (Baron, U. and Bujard, H. Methods Enzymol 327, 401-421 (2000)).A gene encoding bunyavirus (NSm)GnGc is positioned downstream of thetetracycline-responsive element.

In the Tet-Off system, tTA binds to TRE in the absence of TC or dox(Gossen, M. and Bujard, H. Proc Natl Acad Sci USA 89, 5547-5551 (1992))and transcription of bunyavirus (NSm)GnGc proteins is activated, whereastTA cannot bind TRE in the presence of TC or dox and expression isinhibited. In contrast, the Tet-On system uses a reverse tTA (rtTA) thatcan only bind the TRE in the presence of dox (Gossen, M. et al. Science268, 1766-1769 (1995)). Transcription of bunyavirus (NSm)GnGc proteinsis inhibited in the absence of TC or dox and activated in the presenceof TC or dox.

In another embodiment, conditional expression is executed using ahormone inducible gene expression system such as, for instance, anecdysone inducible gene expression system (for example RheoSwitch®, NewEngland Biolabs) (Christopherson, K. S. et al. Proc Natl Acad Sci USA89, 6314-6318 (1992)). Ecdysone is an insect steroid hormone. In cellsexpressing the ecdysone receptor, a heterodimer consisting of theecdysone receptor (Ecr) and retinoid X receptor (RXR) is formed in thepresence of an ecdysone agonist. An exdysone agonist can be selectedfrom ecdysone, one of its analogues such as muristerone A andponasterone A, and a non-steroid ecdysone agonist. In the presence of anagonist, Ecr and RXR interact and bind to an ecdysone response elementthat is present on an expression cassette. Transcription of a proteinthat is placed in an expression cassette downstream of the ecdysoneresponse element is thus induced by exposing the cell to an ecdysoneagonist.

It will be clear to the skilled person that a eukaryotic cell line canbe obtained that expresses sufficient bunyavirus (NSm)GnGc proteins bytransfecting or infecting the eukaryotic cell with a vector thatmediates conditional expression of the bunyavirus (NSm)GnGc proteins.Said vector preferably is a viral vector, for example derived from aparamyxovirus such as a Newcastle disease virus, or, more preferred, anextra chromosomal DNA molecule which is capable of replicatingindependently from the chromosomal DNA such as a plasmid.

In a preferred method according the invention, one or more of thebunyavirus L genome segment, the S genome segment, and/or, when present,the M genome segment comprises a foreign gene. Said foreign gene ispreferably derived from an organism that is a transmitter of aninfectious disease. Said organism is preferably selected fromadenovirus, African horsesickness virus, African swine fever, Bluetonguevirus, Border disease virus, Borna virus, Bovine viral diarrhoe virus,Bovine respiratory syncytial virus, Cache Valley fever virus,Chikungunya virus, Chrysomya bezziana, Classical swine fever,Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus,Cochliomyia hominivorax, Coronavirus, Cytomegalovirus, Dengue virus,Eastern equine encephalitis virus, Ebola virus, Equine encephalomyelitisvirus, Equine encephalosis virus, Foot and mouth disease virus, Goat poxvirus, Hantaanvirus, Hendra virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis E virus, Herpes simplex virus, Highlypathogenic avian influenza virus, Human immunodeficiency virus, Humanparainfluenza virus, Influenza virus, Japanese encephalitis virus,Kaposi's sarcoma-associated herpesvirus, Lassa virus, Lujo virus,Marburg virus, Marsilia virus, Measles virus, Monkeypox virus, Mumpsvirus, Nipah virus, Papillomavirus, Papova virus, Peste des petitsruminants, Polio virus, Polyomavirus, Rabies virus, Respiratorysyncytial virus, Rhinovirus, Rinderpest virus, Rotavirus, Rubella virus,Sandfly fever Naples virus, Sandfly fever Sicilian virus, SARScoronavirus, Sheep pox virus, Simian immunodeficiency virus, Smallpoxvirus, St. Louis encephalitis virus, Toscana virus, Varicella-zostervirus, West Nile virus, Western equine encephalitis virus, Yellow fevervirus, Bacillus anthracis, Bordetella pertussis, Brucella spp.,Campylobacter jujuni, Chlamydia trachomatis, Clostridium botulinum,Coxiella burnettii, Francisella tularensis, Group B streptococcus,Legionella pneumophila, Leptospira spp., Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium ulcerans, Neisseriameningitidis, Salmonella, Shigella spp., Trypanosoma cruzi, Vibriocholerae, Yersinia pestis, Mycoplasma mycoides, Plasmodium malariae,Plasmodium ovale, Plasmodium ssp., Plasmodium vivax, Taenia solium,Taenia spp., and Trypanosoma brucei. Said organism may also be abunyavirus that is the same or different from the bunyavirus from whichthe (NSm)GnGc proteins are derived. Said foreign gene is preferablyderived from an influenza virus and preferably comprises a hemagglutininprotein and/or a neuramidase protein.

In a further preferred method according to the invention, a foreign geneis present on a M, L or S-minigenome. The term “minigenome” refers to anRNA molecule that at least comprises the 5′ and 3′ regions of abunyavirus M, L and/or S genome segment that function in replication andtranscription of the genomic segment. The 5′ and 3′ regions of abunyavirus M, L and/or S genome segment comprise partially complementaryuntranslated regions (UTRs) flanking the coding region of each segment.The terminal 8 nucleotides of these UTRs are conserved between the threesegments, while the remaining sequences of the regions are unique. TheUTRs direct replication and transcription of viral RNA and mediateencapsidation of viral RNA into ribonucleoprotein complexes. Aminigenome is preferably present in addition to a bunyavirus L genomesegment and an S genome segment at least comprising the N-gene. Inaddition, an M genome segment from which the GnGc coding region has beenfunctionally inactivated is optionally present.

When present, the foreign gene is preferably positioned in an expressioncassette that mediates expression of the RNA and/or protein product ofthe foreign gene. It is further preferred that said expression cassettemediates cell-specific or tissue-specific expression of the RNA and/orprotein product of the foreign gene.

The eukaryotic cell in a method of the invention is preferably a cellthat can easily be infected and/or transfected using standard methodsknown to the skilled person, such as, for example, yeast cells andchicken fibroblast cells. Said eukaryotic cell preferably is an insectcell or a mammalian cell. Suitable insect cells comprise, for example,ovarian Spodoptera frugiperda cells such as Sf9 and Sf21, DrosophilaSchneider 2 cells and Aedes albopictus C6/36 cells. Suitable mammaliancells comprise, for example, Baby Hamster Kidney cells such as BHK-21,Human Embryonic Kidney cells such as HEK293, VERO cells, MDCK cells, CHOcells, HuH-7, HeLa, SW13 and PER.C6 cells (Fallaux, F. J. et al. 1998.Hum Gene Ther 9: 1909-1917). A preferred cell is BHK-21.

A method according to the invention can be used to generate arecombinant bunyavirus replicon particle from a bunyavirus that is orwill be known to a skilled person. A method according to the inventionis preferably used to generate a recombinant Crimean-Congo hemorrhagicfever virus replicon particle, a recombinant Nairobi-sheep disease virusreplicon particle, a recombinant Dobrava-Belgrade virus repliconparticle or, most preferred, a recombinant Rift Valley fever virusreplicon particle.

The invention further provides a recombinant bunyavirus repliconparticle, comprising a bunyavirus L genome segment, a bunyavirus Sgenome segment or part of a bunyavirus S genome segment comprising atleast the N gene and, optionally, a bunyavirus M genome segment fromwhich the GnGc coding region has been functionally inactivated. Saidrecombinant bunyavirus replicon particle can be generated using a methodaccording to the invention. Said bunyavirus replicon particle ispreferably selected from the genera Hantavirus, Nairovirus,Orthobunyavirus, and Phlebovirus, which include numerous virus speciescapable of causing severe disease in both animals and humans. Well knownexamples are hantaanvirus (HTNV) and Dobrava-Belgrade virus (DOBV) (bothof the Hantavirus genus), Crimean-Congo hemorrhagic fever virus (CCHFV)and Dugbe virus (both of the Nairovirus genus), Bunyamwera virus(Orthobunyavirus), Oropouche virus (Orthobunyavirus), Rift Valley fevervirus (RVFV, Phlebovirus genus) and further members of the Phlebovirusgenus: Toscana virus, Sandfly fever Naples virus, Punta Toro virus,Uukuniemi virus, Massilia virus and severe fever with thrombocytopeniasyndrome virus. Further preferred bunyaviruses include, but are notlimited to, viruses of the Dera Ghazi Khan virus Group, the Hughes virusGroup, Nairobi sheep disease virus Group, Qalyub virus Group, Sakhalinvirus Group, and the Thiafora virus Group.

It is preferred that one or more of the bunyavirus L genome segment, theS genome segment, and/or, when present, the M genome segment ofrecombinant bunyavirus replicon particle according the invention,comprises a foreign gene. Said foreign gene is preferably derived froman organism that is a transmitter of an infectious disease. As analternative, a preferred recombinant bunyavirus replicon particleaccording to the invention comprises a foreign gene that is present on aM, L or S-minigenome.

A preferred recombinant bunyavirus replicon particle according to theinvention is derived from a bunyavirus selected from Crimean-Congohemorrhagic fever virus, Nairobi-sheep disease virus, Dobrava-Belgradevirus and Rift Valley fever virus. A most preferred bunyavirus is RiftValley fever virus.

The invention additionally provides a method for producing a recombinantbunyavirus replicon particle, the method comprising A) providing aeukaryotic cell with growth medium, B) providing the eukaryotic cellwith sufficient bunyavirus (NSm)GnGc, and C) infecting the eukaryoticcell with a recombinant bunyavirus replicon particle according to theinvention, so as to produce a bunyavirus replicon particle. Saideukaryotic cell is preferably a cell that is persistently infected witha viral vector that transduces the bunyavirus (NSm)GnGc proteins, or astable cell in which an expression construct that expresses theBunyavirus (NSm)GnGc proteins is integrated into the genome. Saidexpression construct preferably comprises means for conditionalexpression of the bunyavirus (NSm)GnGc proteins.

A recombinant bunyavirus replicon particle according to the invention issafe and can be used outside a biosafety containment. Said recombinantbunyavirus replicon particle can be used, for example, for screening anddevelopment of anti-viral agents, for example for development of a highthroughput system for screening of suitable compound libraries. Saidrecombinant bunyavirus replicon particle can also be used in tests orassays including virus neutralization assays or virus neutralizationtests (VNT) and ELISAs, including whole-virus ELISAs, andhemagglutination assays.

For example, a classical VNT requires handling of live bunyavirus andmust therefore be performed in appropriate biosafety containmentfacilities. Another drawback of the classical VNT is that the assayrequires 5-7 days for completion. An advantage of the use of bunyavirusreplicon particles, such as RVFV replicon particles (RRPs), in stead oflive bunyavirus, is that the VNT can be performed outsidebio-containment facilities. A further advantage is that the VNT requiresonly 24-48 hrs for completion.

The invention further provides a recombinant bunyavirus repliconparticle according the invention for use as a medicament. A recombinantbunyavirus replicon particle according the invention is preferably foruse as a medicament for amelioration of a bunyavirus infection in ananimal, including in a human.

A pharmaceutical medicament comprising a recombinant bunyavirus repliconparticle according the invention may additionally comprise apharmaceutical acceptable adjuvant, diluent or carrier. A medicamentaccording to the invention is preferably combined with other therapeuticoptions, including but not limited to a combination treatment withribavirin, and/or derivatives of ribavirin such as taribavirin.

The invention further provides a vaccine comprising a recombinantbunyavirus replicon particle according to the invention. Said vaccinepreferably comprises an adjuvant. Adjuvant substances are used tostimulate immunogenicity. Examples of commonly used immunologicaladjuvants are aluminum salts, immunostimulating complexes (ISCOMS),non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2,IL-7, etc.), saponins, monophosphoryl lipid A (MPLA), muramyldipeptides, vitamin E, polyacrylate resins, and oil emulsions.Preferably, the adjuvant is a sulfolipopolysaccharide, such as theSLP/S/W adjuvant described in Hilgers et al. Vaccine 1994 12:653-660. Afurther preferred adjuvant is provided by a triterpene, such assqualene, and derivatives and modifications thereof.

Bunyavirus replicon particles, for example RRPs, according to theinvention are non-spreading particles that are capable of autonomousgenome replication. Bunyavirus replicon particles according to theinvention contain both the S and L genome segment, or functional partsthereof, but lack a bunyavirus M genome segment or comprise a bunyavirusM genome segment on which the GnGc coding region has been functionallyinactivated. The presence of the L and S genome segments, which encodethe N protein and the L protein, enables the resulting particles ofautonomous genome replication. The absence or inactivity of a GnGccoding region prevents assembly and spread of virus particles. Thereplicon particles can be produced in cells that supply GnGc in trans totiters up to 10E7 infectious particles/ml. Replication of the bunyavirusgenome and/or expression of the N and L protein in a host might resultin an improved immune response, when compared with an immune responseinduced by vaccination with bunyavirus-like particles or viral subunits.For example, a single intramuscular vaccination with RRPs protects miceagainst a lethal challenge dose with RVFV strain 35/74.

Said vaccine may be administered to an animal, including a human, by anymethod known in the art. Said vaccine is preferably administered byneedle-free, non-invasive methods such as oral, intranasal and/orintratracheal administration, for example through inhalation or the useof nose-sprays. Said vaccine is more preferably parenterallyadministered, for example, intramuscularly, subcutaneously,intraperitoneally, intradermally or the like, preferablyintramuscularly.

A vaccine according to the invention is administered in effectiveamounts according to a schedule which may be determined by the time ofanticipated potential exposure to a bunyavirus. In this way, the treatedanimal, including a human, may have time to build immunity prior to thenatural exposure. A typical treatment schedule or dosing regimencomprises parenteral administration, preferably intramuscular injection,of one dosage unit, at least about 2-8 weeks prior to potentialexposure. If required, a second dosage unit is administered at about 2-4weeks prior to potential exposure. The second dosage may be administeredby the same method, or by a method differing from the first dosage unit.

The administration of a vaccine according to the invention preferablyprotects the animal, including a human, against a subsequent infectionby the bunyavirus. In a preferred embodiment, a vaccine according to theinvention comprises a recombinant Rift Valley fever virus repliconparticle that protects the animal, including a human, against asubsequent infection by Rift Valley fever virus.

An important advantage of a vaccine according to the invention is thatthe virus is not capable of autonomous spread in the vaccinated animal.The inability to spread from the initial site of inoculation, greatlyadds to the safety of this vaccine, both for the inoculated animal, theadministrator of the vaccine as well as the environment. The inabilityto cause viremia in the vaccinated animal also prevents any concernsabout possible transmission of this vaccine by insect vectors (seeMoutailler et al. 2010. Vector Borne Zoonotic Dis 10:681-688). A vaccinecomprising a non-spreading Bunyavirus replicon particle according to theinvention which lacks the NSs gene (such as those reported in Example 1and in Kortekaas et al. 2011. J. Virol. Accepted for publication) areconsidered of optimal safety, due to the lack of this major virulencefactor.

A further important advantage of a vaccine according to the inventioncompared to inactivated vaccines or subunit vaccines is the fact thatthe present vaccine does not depend on an adjuvant to induce immunity.In addition, a vaccine according to the invention can be produced withsuperior cost-effectiveness and will be superior with respect toduration of immunity. Although the virus is non-spreading, due to theinability to express GnGc protein, the viral genome is replicated ininfected cells and N- and L-proteins are expressed, resulting in astrong, long-lasting induction of an immune response in a recipient.

It is important to note that there are concerns about the safety of theMP-12 (Morrill et al. 1991. Vaccine 9:35-41, Morrill et al. 1997. Am JVet Res 58:1104-1109, Morrill et al. 1997. 58:1110-1114, Morrill et al.2011. 204:229-236, Morrill et al. 2011. J Infect Dis 204:617-625), Clone13 (Muller et al., 1995. Am J Trop Med Hyg. 53:405-411, Vialat et al.2000. J Virol 74:1538-1543, Dungu et al. 2010. Vaccine 28:4581-4587) andR566, a reassortant virus that contains the S segment of the Clone 13virus and the L and M segments of the MP-12 virus (Flick et al. 2009.Antiviral Res 84:101-118) vaccine viruses. The MP-12 vaccine virus wasshown to contain potential attenuating mutations on each of the threegenome segments (Vialat et al. 1997. Virus Res 52:43-50). The nucleotidechanges responsible for attenuation of this virus are, however, notmapped. It is therefore possible that only a single nucleotide changecould result in reversion to virulence. Although several studies havedemonstrated the safety of the MP-12 vaccine (see papers of Morrill etal. noted above), a further study demonstrated that the MP-12 vaccine isnot safe when administered to gestating ewes during the first trimesterof gestation (Hunter et al., 2002 Onderstepoort J Vet Res 69; 95-98).Since the R566 vaccine contains the L and M segments of the MP-12 virus,similar concerns may be raised about the safety of this vaccine.

Death of Clone 13-vaccinated mice due to neurological disorders andparalysis was reported in one of the first articles on the Clone 13virus (Vialat et al. 2000. J Virol 74:1538-1543). This finding suggestedthat the safety of the Clone 13 virus should also be further studied inclinical trials involving large numbers of animals.

None of the animals that were vaccinated with a Bunyavirus repliconparticle according to the invention suffered from complicationsassociated with the vaccination.

The invention further provides a method of stimulating an immuneresponse against bunyavirus in an animal, including a human, the methodcomprising providing the animal with a recombinant bunyavirus repliconparticle according to the invention. In a preferred method, arecombinant Rift Valley fever virus replicon particle according to theinvention is provided to an animal, including a human, to stimulate animmune response against Rift Valley fever virus.

The invention further provides the use of a recombinant bunyavirusreplicon particle according to the invention, for stimulating an immuneresponse against a protein encoded by the foreign gene. In oneembodiment, said foreign gene preferably encodes an antigenic proteinthat is expressed by an Orthomyxovirus, preferably influenza A virus, oran immunologically-active part or derivative of a protein that isexpressed by an Orthomyxovirus. Methods for determining whether aprotein, or a part or derivative of a protein, is immunologically activeare known to the person skilled in the art, including algorithms thatpredict the immunogenicity of a protein such as an algorithm of Parkerand an algorithm of Rammensee, as disclosed in Provenzano et al. 2004.Blood 104: Abstract 2862) and including the injection of the purifiedprotein, or a part or derivative of the protein in a suitable animal anddetermining whether the protein, or a part or derivative of a protein iscapable of stimulating antibodies against the protein, or a part orderivative of a protein.

The term immunologically-active part indicates a part of a protein thatis able to induce a cellular and/or humoral immune response against theprotein in an animal, including a human. The term immunologically-activederivative indicates a protein or part of a protein that is modified,for example by addition, deletion or alteration of one or more aminoacids and which is able to induce a cellular and/or humoral immuneresponse against the protein in an animal, including a human. It ispreferred that an immunologically-active derivative has a sequenceidentity of more than 70% compared to the parental protein, for examplethe protein that is expressed by the Orthomyxovirus, preferably aninfluenza A virus. The sequence identity is more preferred more than80%, more preferred more than 90%, more preferred more than 95%, morepreferred more than 99%, most preferred 100%, as based on the amino acidsequence of the protein or protein parts. Said immunologically-activederivative is, for example, a protein that comprises a signal peptidefor secretion out of the cell in which it is produced, a protein thatcomprises a sequence that provides a trans-membrane such as a type I, IIor III targeting domain, or a protein in which a protease cleavage sitehas been altered to enhance the half-life of the protein.

FIGURE LEGENDS

FIG. 1.

Expression of the N protein from the RVFV S genome segment. BSR-T7/5cells (A) or FP-T7-infected BHK-21 cells (B) were transfected withplasmid pUC57-S, encoding the RVFV S genome segment in theantigenomic-sense orientation. Expression of the RVFV N protein wasdetected using a N protein-specific mAb and HRP-conjugated anti-mouseIgG antibodies.

FIG. 2.

Production of Rift Valley fever virus replicon particles (RRPs)containing three genome segments. BHK cells were infected with FP-T7 andsubsequently transfected with a plasmid encoding the M-eGFP minigenome(M-eGFP), or in combination with plasmids encoding the RVFV L and Sgenome segments (M-eGFP/L/S), or in combination with the aforementionedplasmids and pCAGGS-NSmGnGc (M-eGFP/L/S+NSmGnGc). The number ofeGFP-positive producer cells, recipient cells or recipient cellspreviously transfected with helperplasmids (+HP) pCIneo-RVFV-L andpCAGGS-N were determined by flow cytometry.

FIG. 3.

Production of RRPs containing two genome segments. BHK cells wereinfected with FP-T7 and subsequently transfected with a plasmid encodingthe S-eGFP minigenome (S-eGFP), or in combination with plasmids encodingthe RVFV L genome segments (S-eGFP/L), or in combination with theaforementioned plasmids and pCAGGS-NSmGnGc (S-eGFP/L+NSmGnGc). Thenumber of eGFP-positive producer cells, recipient cells or recipientcells previously transfected with helperplasmids (+HP) pCIneo-RVFV-L andpCAGGS-N were determined by flow cytometry.

FIG. 4.

Expression of GnGc in BHK-GnGc cells. Detection of GnGc expression inBHK-21 cells (negative control, A) and BHK-GnGc cells (B) by IPMA usingpolyclonal antibodies directed against the Gn and Gc proteins anddetection of Gc in cell lysates on a Western blot (C) using a peptideantiserum.

FIG. 5.

Expression of eGFP in BHK-GnGc cells persistently infected with NDV-GnGcmaintaining the RVFV L genome segment and S-eGFP minigenome at cellpassage 18.

FIG. 6.

RRP production by BHK-GnGc cells after introduction of pCAGGS-GnGc orpCAGGS-NSmGnGc. At 16 h after transfection, the culture medium wasreplaced by fresh medium. RRP titers were determined on BHK-21 cellsusing the Spearman-Kärber method (Kärber 1931. Arch. Exp. Path. Pharmak162, 480-483; Spearman 1908 Br. J. Psychol 2, 227-242).

FIG. 7.

(A) Partial sequence pUC57-L (SEQ ID NO:20): Plasmid encodes the RVFVstrain 35/75 L genome segment in antigenomic-sense orientation.

cDNA of the RVFV strain 35/74 L genome segment, flanked by a T7 promoterand cDNA encoding a HDV ribozyme was synthesized by the GenScriptCorporation and cloned in the pUC57 vector using KpnI/HindIII. The T7promoter, HDV ribozyme sequence and T7 terminator sequences,respectively, are underlined. The UTRs are indicated in italics, theopen reading frame encoding the L protein is indicated in bold.

(B) Partial sequence of plasmid pUC57-M (SEQ ID NO:21): Plasmid encodesthe RVFV strain 35/74M genome segment in antigenomic-sense orientation.

eDNA encoding the antigenomic-sense RNA of the RVFV strain 35/74 Mgenome segment, flanked by a T7 promoter and cDNA encoding a HDVribozyme was synthesized by the GenScript Corporation and cloned in thepUC57 vector using EcoRI/SalI. The T7 promoter, HDV ribozyme sequenceand T7 terminator sequences, respectively, are underlined. The UTRs areindicated in italics, the open reading frame encoding the NSmGnGcproteins is indicated in bold.

(C) Partial sequence of plasmid pUC57-S(−) (SEQ ID NO:22): Plasmidencodes the RVFV strain 35/74 S genome segment in genomic-senseorientation.

cDNA encoding the genomic-sense RNA of the RVFV strain 35/74 S genomesegment, flanked by a T7 promoter and cDNA encoding a HDV ribozyme wassynthesized by the GenScript Corporation and cloned in the pUC57 vectorusing BamHI/XbaI. The T7 promoter, HDV ribozyme sequence and T7terminator sequences, respectively, are underlined. The UTRs andintergenic region are indicated in italics, the open reading framesencoding the N and NSs proteins are indicated in bold.

(D) Partial sequence of plasmid pUC57-SANSs (SEQ ID NO:23): Plasmidencodes the RVFV strain 35/74 S genome segment in antigenomic-senseorientation with a major deletion in NSs.

The synthetic DNA was cloned between XbaI and ApaI of pUC57. The T7promoter, HDV ribozyme sequence and T7 terminator sequences,respectively, are underlined. The UTRs and intergenic region areindicated in italics, the open reading frame encoding the N and the openreading frame encoding part of the NSs protein are indicated in bold.

(E) Partial sequence of plasmid pUC57-S (SEQ ID NO:24): Plasmid encodesthe RVFV strain 35/74 M genome segment in antigenomic-sense orientation.

To construct cDNA encoding the complete S genome segment inantigenomic-sense orientation, the sequence between NcoI, EcoRV wasisolated from the pUC57-S(−) construct and used to replace the sequencebetween NcoI and EcoRV of plasmid pUC57-SΔNSs, yielding pUC57-S. The T7promoter and HDV ribozyme and T7 terminator sequences, respectively areunderline. The UTRs and intergenic region are indicated in italics, theopen reading frames encoding the N and NSs proteins are indicated inbold.

(F) Partial sequence of plasmid pUC57-S-eGFP (SEQ ID NO:25): Plasmidencodes the RVFV strain 35/74 NI genome segment in antigenomic-senseorientation, where the NSs gene is replaced for the gene encodingenhanced green-fluorescent protein (eGFP).

The T7 promoter and HDV ribozyme and T7 terminator sequences,respectively are underlined. The UTRs and intergenic region areindicated in italics, the open reading frames encoding the N and eGFPproteins are indicated in bold. The sequence was cloned between KpnI andSalI of pUC57. A silent C→T mutation is underlined.

(G) Partial sequence of plasmid pUC57-Mv (SEQ ID NO:26): Plasmid encodesthe RVFV strain 35/74M genome segment in genomic-sense orientation,where the complete NSmGnGc ORF is deleted and NcoI and XbaI sites areintroduced.

The T7 promoter and HDV ribozyme and T7 terminator sequences,respectively are underlined. The UTRs are indicated in italics, thesequence between the UTRs is indicated in bold. Cloned between EcoRI andPstI pUC57.

(H) Partial sequence of plasmid pUC57-Mv-eGFP (SEQ ID NO:27): Plasmidencodes the RVFV strain 35/74 M genome segment in genomic-senseorientation, where the complete NSmGnGc ORF is deleted and eGFP isintroduced between the NcoI and XbaI sites. The T7 promoter and HDVribozyme sequences are underlined.

(I) Partial sequence of plasmid pUC57-GnGc (SEQ ID NO:28): Plasmidencodes a codon-optimized version of the open reading frame of the RVFVstrain 35/74 M genome segment starting at the fourth methionine codon.The gene was synthesized and cloned between EcoRI and HindIII. This genewas used to construct pCIneo-GnGc and pCAGGS-GcGc Using EcoRI and NotI(underlined). The GnGc open reading frame is indicated in bold.

(J) Plasmids pCIneo-NSmGnGc and pCAGGS-NSmGnGc encode the open readingframe of the RVFV strain 35/74 M genome segment starting at the firstmethionine codon (SEQ ID NO:29). The gene was amplified from 35/74 cDNAand cloned in pCIneo and pCAGGS using EcoRI and NotI (underlined). TheNSmGnGc open reading frame is indicated in bold.

(K) Plasmid pUC57-N encodes a codon-optimized version of the N openreading frame of the RVFV strain 35/74 (SEQ ID NO:30). The gene wasintroduced into pCAGGS and pCIneo using EcoRI and NotI (underlined). Theopen reading frame of the N protein is indicated in bold.

(L) Plasmid pCIneo-L contains the open reading frame of the L gene ofRVFV strain 35/75 (SEQ ID NO:31). The gene was introduced into usingXhoI and NotI (underlined). The transition mutation (T5912C), resultingin the substitution of isoleucin-1971 for threonine is also underlined.The open reading frame of the L protein is indicated in bold.

FIG. 8.

RRPs are incapable of autonomous spread. BHK cells were infected withRRPs at an m.o.i. of 1. After two days, eGFP expression was observed ininfected cells (left panel). Fresh BHK cells were incubated with thecollected supernatant and monitored for eGFP expression after three days(right panel).

FIG. 9.

RRP production kinetics. BHK-Rep cells were grown in GMEM supplementedwith 5% serum and were either left untreated (−GP) or transfected withpCAGGS-NSmGnGc (+GP). RRP titers were determined at different timepoints on BHK cells using eGFP expression as the readout parameter.Titers were determined using the Spearman-Kärber method.

FIG. 10.

Western blot analysis of RRP proteins. Culture medium of BHK-Rep cells(−GP) or of BHK-Rep cells transfected with pCAGGS-NSmGnGc (+GP) wasultracentrifuged at 100 000×g for 2 h. The proteins present in thepellets were separated in 4-12% NuPAGE Bis-Tris gels and subsequentlytransferred to nitrocellulose blots. Specific proteins were detected byan anti-Gn (α-Gn) or anti-Gc (α-Gc) peptide antiserum or a mAb specificfor the N protein (αN). The positions of the NSm, Gn, Gc and N proteinare indicated by arrows. Molecular weight standard proteins areindicated to the right in kilodaltons.

FIG. 11.

RRP infection of mammalian and insect cells. Cells were infected withRRPs and the number of positive cells was determined by flow-cytometryat 42 (BHK and HEK293T) or 72 hours post infection (S2 and C6/36). (A)Representative result from flow cytometry of RRP-infected mammalian andinsect cells. Counts of non-infected control cells and infected cellsare depicted in grey and green respectively. (B) Histograms showingaveraged results of three independent measurements with S.D.

FIG. 12.

Vaccine efficacy of RRPs. Mice were either non-vaccinated (n=9; Mock) orvaccinated (n=10) either once (1×) or twice (2×) via the intramuscularroute (IM) or subcutaneous route (SC) with 106 TCID50 of RRPs. Mice werechallenged with a known lethal dose of RVFV strain 35/74 via theintraperitoneal route. The mortality rates were determined until 21 dayspost challenge (d.p.c.).

FIG. 13.

Injection site reactions observed at different time points aftervaccination. Score 0: No aberrations noted; 1: Swelling observed; 2:Round swelling of maximal 5 cm in diameter; 3: Major swelling/abscesschance of rupture. PM, post mortem. Results are depicted as averages(n=6) with SD.

FIG. 14.

Rectal temperatures of vaccinated and unvaccinated (Mock) lambs beforeand after challenge with RVFV. Rectal body temperatures (° C.) weredetermined daily. Fever was defined as a body temperature above 41° C.(interrupted line). Results are depicted as averages (n=6) with SD.

FIG. 15.

Detection of viral RNA in plasma samples of vaccinated and unvaccinatedlambs obtained at different time points after challenge infection withRVFV. The number of positive samples differs at each time point. Resultsare depicted as averages (n=6) with SD.

FIG. 16.

Body weight of lambs vaccinated once with one of the indicated vaccines.Results are depicted as averages (n=6) with SD.

FIG. 17.

Biochemical analysis of serum samples. ALT, ALP, TP, Creatinine and BUNconcentrations are depicted as averages (n=6) with SD. The upper andlower reference values are indicated by dashed lines.

FIG. 18.

Schematic representation of the method used to create replicon celllines producing the sHA₃ and sNA₄ proteins. GOI, gene of interest (inthis work either the eGFP, sHA₃ or sNA₄, gene).

FIG. 19.

Flow cytometry demonstrating the percentage of replicon cell lines thatproduce the N protein at cell passage 8. N protein expression isdependent on the presence of the S and L genome segment, of which theformer contains the foreign gene of interest (GOI). Cells expressing Land S-eGFP genome segments (A); cells expressing L and theS-CD5-HA-GCN4-ST (i.e. S-_(s)HA₃) segment (B); cells expressing L andthe S-CD5-OS-GCN4-NA segment (i.e. 5-_(s)NA₄) (C); control cellsexpressing GnGc are depicted in (D), (E) and (F).

FIG. 20.

Purified sHA₃ and sNA₄ were analyzed on Silver-stained polyacrylamidegels (A) and Western blots using Strep-Tacting-HRP for detection (B).The negative control sample (eGFP) was provided by following thepurification procedure using medium collected from replicon cellsexpressing eGFP. The positions of molecular weight standard proteins areindicated to the left in kDa.

EXAMPLES Example 1 Materials and Methods

Cells and Growth Conditions.

BSR-T7/5 were kindly provided by Prof. Dr. K. Conzelmann (Max vonPettenkofer-Institut, Munchen, Germany). BHK-GnGc are BHK-21 cells thatcontain a genome-integrated plasmid pCIneo-GnGc described hereinbelow.BSR-T7/5 and BHK-GnGc cells were grown in Glasgow Minimum EssentialMedium (GMEM; Invitrogen, Carlsbad, Calif., USA) supplemented with 4%tryptose phosphate broth (Invitrogen), 1% non-essential amino acids(Invitrogen), 10% fetal bovine serum (FBS; Pam Biotech, Aidenbach,Germany) and penicillin/streptomycin (Invitrogen) at a concentration of100 U/ml and 100 μg/ml, respectively. For maintenance of stable celllines, geneticin (G-418; Invitrogen) was used at a concentration of 1mg/ml. Cells were grown at 37° C. and 5% CO2.

Plasmids and Viruses.

Plasmids pCIneo-GnGc and pCAGGS-GnGc contain the open reading frame(ORF) of the M segment of RVFV strain 35/74, starting at the fourthmethionine codon (FIG. 7I). Plasmid pCAGGS-N contains the ORF encodingthe N gene (FIG. 7K). The N and GnGc-encoding sequences werecodon-optimized for optimal expression in mammalian and insect cells andsynthesized by the GenScript Corporation (Piscataway, N.J., USA).Plasmid pCAGGS-NSmGnGc contains the authentic ORF of the M segmentstarting at the first methionine codon (FIG. 7J). pCIneo-RVFV-L encodesthe authentic ORF encoding the viral polymerase of RVFV strain 35/74(FIG. 7L). This gene contains a transition mutation (T5912C), resultingin the substitution of isoleucin-1971 for threonine. The effect of thismutation was not studied. Expression of RVFV genes from pCIneo plasmidsis controlled by a cytomegalovirus (CMV) immediate-earlyenhancer/promoter, whereas the expression of genes from pGAGGS plasmidsis controlled by a CMV immediate enhancer/β-actin (CAG) promoter (Niwaet al. 1991. Gene 108: 193-199). RVFV strain 35/74 was isolated at theAgricultural Research Council-Onderstepoort Veterinary Institute(ARC-OVI) from the liver of a sheep that died during a RVFV outbreak inthe Free State province of South Africa in 1974 (Barnard 1979. J S AfrVet Assoc 50: 155). The virus was passaged four times in suckling miceby intra-cerebral injection and three times on BHK-21 cells.Amplification of the genome segments was performed by one-step RT-PCRwith SBS Genetech AMV-RT, TaKaRa Ex Taq HS and the primers described byBird et al. (Bird et al. 2007. J Virol 81: 2805-2816). PCR products werepurified with the Qiagen Gel extraction kit after separation on agarosegel and mixed in appropriate equal amounts before GS FLX sequencing atInqaba Biotec (Pretoria, South Africa). Sequencing and sequence assemblywas performed essentially as described for dsRNA virus genomes(Potgieter et al. 2009. J Gen Virol 90: 1423-1432). Consensus sequencescorresponding to each genome segment were synthesized and cloned inpUC57, a standard cloning vector of GenScript Corporation (Piscataway,N.J., USA). pUC57-L (FIG. 7A), pUC57-M (FIG. 7B) and pUC57-S (FIG. 7E)encode the RVFV L, M and S genome segment in antigenomic (i.e. positive)sense orientation, respectively. These transcription plasmids eachcontain a complete copy of the viral RNA segments and are flanked by aT7 promoter and a HDV ribozyme sequence. In pUC57-S-eGFP(−) (FIG. 7F),the NSs gene is replaced by the gene encoding enhanced green fluorescentprotein (eGFP). Of note, transcription of this plasmid results in aS-eGFP minigenome in which the eGFP gene is in the genomic (i.e.negative-sense) orientation. In plasmid pUC57-Mv-eGFP(−) (FIG. 7H), thecomplete ORF of the M segment is replaced by the eGFP gene. The M-eGFPminigenome produced from this plasmid is in the genomic-senseorientation as well.

A recombinant Newcastle disease virus (NDV) that produces the RVFVstructural glycoproteins Gn and Gc (i.e. NDFL-GnGc), from hereonreferred to as NDV-GnGc, was previously described (Kortekaas et al.2010. Vaccine 28: 4394-4401).

A recombinant fowlpox virus that produces T7 polymerase, namedfpEFLT7pol (Das et al. 2000. J Virol Meth 89: 119-127), from hereonreferred to as FP-T7, was kindly provided by the Institute for AnimalHealth (IAH, Compton, UK). Virus titers were determined as 50% tissueculture infective dose (TCID50) on BHK-21 cells using theSpearman-Kärber method (Kärber 1931. Arch. Exp. Path. Pharmak 162,480-483; Spearman 1908 Br. J. Psychol 2, 227-242),

Rescue of Recombinant RVFV Strain 35/74.

BSR-T7/5 cells were seeded in 6-well plates and were co-transfected with1 μg of plasmids pUC57-L, pUC57-M and pUC57-S using jetPEI transfectionreagent according to the instructions of the manufacturers(Polyplus-transfection SA, Illkirch, France). After 6 days ofincubation, medium was collected. For the detection of infectious virus,BHK-21 cells were incubated with the collected supernatant. When clearcytopathic effect was observed, the cells were fixed in 4%paraformaldehyde/PBS for 40 min. Plates were subsequently submergedcompletely in 80% ethanol/4% acetic acid to inactivate the virus andwashed with PBS. Immunoperoxidase monolayer assays (IPMAs) wereperformed as described hereinbelow.

Alternatively, BHK-21 cells were infected with FP-T7. Approximately 10E6cells in each well of a six-well plate were inoculated with 1.5 ml ofculture medium containing 10E5 TCID50 of FP-T7 (multiplicity ofinfection [m.o.i.] of 0.1). After incubation with FP-T7 for 1 h andrecovery for another hour, the cells were treated in a similar way asdescribed for BSR-T7/5 cells.

Rescue of RVFV BRPs (RRPs).

BHK-21 or BHK-GnGc cells were seeded in 6-well plates and incubated withFP-T7 for 1 h at 37° C. Medium was refreshed and cells were allowed torecover for 1 h. For the production of RRPs containing three genomesegments, the cells were subsequently transfected with 600 ng ofplasmids pUC57-L, pUC57-S, pUC57-Mv-eGFP(−) and pCAGGS-NSmGnGc. For theproduction of RRPs containing two genome segments, cells weretransfected with pUC57-L, pUC57-S-eGFP(−) and pCAGGS-NSmGnGc. The mediumwas refreshed the next day. Alternatively, when NDV was used to provideGn and Gc, NDV-GnGc infection was performed together with FP-T7.NDV-GnGc was used at an m.o.i. of 0.05. Supernatants were harvestedafter 72 h, pre-cleared at 5 000 rpm for 5 min at RT and stored at 4° C.until further use.

NuPAGE and Western Blotting

NuPAGE and Western blotting was performed as described (Kortekaas et al.2010. Vaccine 28: 2271-2276). Briefly, proteins were diluted in 3×Laemmli sample buffer (0.5 M Tris pH 6.8, 6% (w/w) SDS, 26% (v/v)glycerol, 15% (v/v) 2-mercaptoethanol and 0.002% (w/w) bromophenol blue)and heated at 95° C. for 5 minutes, before loading onto 4-12% NuPAGEBis-Tris gels. Proteins were subsequently transferred to nitrocelluloseblots. To visualize Gc, rabbit polyclonal antibodies were used that werepreviously raised against a Gc-derived peptide (residues975-VFERGSLPQTRNDKTFAASK-994), respectively (De Boer et al. 2010.Vaccine 28:2330-2339). Goat anti-rabbit IgG antibodies conjugated tohorseradish peroxidase (HRP) (Dako, Heverlee, Belgium) were used as thesecondary antibodies. Peroxidase activity was detected using theAmersham ECL™ Western blotting detection reagents (GE Healthcare,Diegem, Belgium). For the detection of the N protein, monoclonalantibody F1D11 (kindly provided by Dr. Alejandro Brun, CISA-INIA,Madrid, Spain) was used as the primary antibody and rabbit anti-mouseIgG (DAKO) as the secondary antibody.

Immunoperoxidase Monolayer Assays (IPMA).

IPMAs were performed as described previously (deBoer et al. 2010.Vaccine 28: 2330-2339). As the primary antibody, either a polyclonalantiserum was used that was previously obtained from a sheep that wasvaccinated with NDFL-GnGc (Kortekaas et al. 2010. Vaccine 28:4394-4401). For detection of the RVFV N protein, mAb F1D11 was used,which was previously kindly provided by Dr. Alejandro Brun (CISA-INIA,Spain). As the secondary antibody HRP-conjugated rabbit anti-mouse IgG(Dako, Heverlee, Belgium) was used.

Flow Cytometry.

For flow cytometry, cells of a six-well plate were washed with 3 ml PBSand then trypsinized with 0.3 ml 0.5% trypsine-EDTA (Invitrogen). Afterincubation for 2-3 min at 37° C. and resuspension, the cells werediluted in 1 ml culture medium. Cells were pelleted by centrifugation,resuspended in 0.5 ml PBS and pelleted again. Cells were fixed by adding0.1 ml 4% PFA in PBS for 15-30 min and subsequently diluted in 0.5 mlPBS. Each sample contained all the cells from one well. The samples werestored at 4° C. until analysis. All measurements were performed at theday of harvesting the cells. Flow cytometry was performed using a CyAnADP flow cytometer (Beckman, Woerden, The Netherlands), equipped with a488 nm wavelength laser. Data analysis was performed with the Summitv4.3 software.

Results

Rescue of recombinant RVFV strain 35/74 from cDNA using BSR-T7/5 cells.Recombinant RVFV strain 35/74 (rec35/74) was rescued using threeplasmids encoding the three viral RNA segments L, M and S inantigenomic-sense orientation. Transfection of the three plasmids intoBSR-T7/5 cells resulted in cytopathic effect after 3-4 days. BHK-21cells were inoculated with the collected supernatant. A titer of 10E9TCID50/ml was obtained after two passages of the virus. No silentmutations were introduced in the genome of recombinant RVFV strain 35/74to confirm rescue and exclude contamination with non-recombinantwildtype virus, since no wildtype virus was ever present in ourlaboratory before rescue of rec35/74.

Production of VLPs containing a reporter minigenome using transientexpression of NSmGnGc from plasmid.

As a first step towards the establishment of an Rift Valley fever virusreplicon particle (RRP) production system, a minireplicon system wasdeveloped. A plasmid was designed in which the gene encoding eGFP isplaced between M segment untranslated regions (UTRs). This plasmid wasnamed pUC57-Mv-eGFP(−) and encodes the M-eGFP minigenome ingenomic-sense orientation. Like the full-length constructs, the cDNAencoding this minigenome is flanked by a T7 polymerase promoter and cDNAencoding a HDV ribozyme sequence. Transfection of pUC57-Mv-eGFP(−)together with plasmids pUC57-L and pUC57-S resulted in eGFP expressionin only a few cells (data not shown). In an attempt to improve reporterminigenome expression, the two helper plasmids pCIneo-RVFV-L andpCAGGS-N were added to the transfection mixture. This resulted in aslight increase in the number of positive cells (data not shown).Despite the very low number of eGFP-positive cells, an attempt was madeto package the genome segments into VLPs by co-transfecting the cellswith pCAGGS-NSmGnGc. If indeed VLPs were produced, we reasoned thatthese VLPs could contain either one, two or three genome segments. To beable to detect also VLPs that contain only the reporter minigenome, thesupernatant was not only added to untreated BHK-21 cells, but also toBHK-21 cells that were previously transfected with helper plasmidspCIneo-RVFV-L and pCAGGS-N. Very few BHK-21 cells that were inoculatedwith the collected supernatant were shown to express eGFP after 18-24hrs of incubation and expression was only observed in cells that werepreviously transfected with helper plasmids (data not shown). Thus,although VLPs were produced, none of these VLPs apparently contained allthree genome segments.

Production of VLPs containing a reporter minigenome using NDV to provideGn and Gc.

Although we were previously able to produce large amounts of VLPs byexpressing GnGc in insect cells (de Boer et al. 2010. Vaccine 28:2330-2339), production of GnGc from pol-II promoters in mammalian cellswas extremely poor in previous experiments, yielding no detectable VLPsin the culture supernatant (unpublished results). We also previouslyreported the production of an NDV recombinant (i.e. NDFL-GnGc, fromhereon referred to as NDV-GnGc) that produces the GnGc glycoproteins.Interestingly, infection of BHK-21 cells with this recombinant virus didresult in the production of detectable amounts of Gn and Gc in thesupernatant (Kortekaas et al. 2010. Vaccine 28: 4394-4401). In thepresent work, we wondered if NDV-GnGc could be used to provide the Gnand Gc proteins for the packaging of RVFV genome segments. Cells werefirst transfected with pUC57-L, pUC57-S and pUC57-Mv-eGFP(−) andinfected with NDV-GnGc, 24 hrs later. Expression of eGFP was observed ina small percentage of producer cells and in very few recipient cells(data not shown). Expression of eGFP in recipient cells was againdependent on a previous transfection with the helper plasmidspCIneo-RVFV-L and pCAGGS-N. These experiments demonstrated that we wereable to package a reporter minigenome into VLPs using NDV-GnGc as asource of the glycoproteins, but that particles capable of autonomousreplication were not obtained.

Improved rescue of RVFV from cDNA using a recombinant fowlpox virus as asource of T7 polymerase and successful production of RRPs.

Virus recovery from BSR-T7/5 cells was not reproducible. As analternative for BSR-T7/5 cells, we decided to use a recombinant fowlpoxvirus that expresses T7 polymerase (i.e. FP-T7) (Das et al. 2000. JVirol Methods 89: 119-127). In experiments where the rescue efficiencyof RVFV using BSR-T7/5 cells and FP-T7-infected BHK-21 cells werecompared, use of FP-T7 resulted in RVFV rescue in 5/5 attempts, whereasrescue using BSR-T7/5 cells was unsuccessful. We anticipated that ahigher level of T7 polymerase expression by FP-T7 could result in higherproduction levels of the N and L proteins from the antigenomic (i.e.positive-sense) genome segments, facilitating initiation of replication.To test this hypothesis, the pUC57-S segment was transfected intoBSR-T7/5 cells and into BHK-21 cells that were previously infected withFP-T7. Transcription of the pUC57-S plasmid by T7 polymerase results inantigenomic sense viral RNA of which the N gene is in sense orientation.Whereas the N protein could not be detected in BSR-T7/5 cellstransfected with pUC57-S (FIG. 1A), FP-T7-infected BHK-21 cells thatwere transfected with this plasmid stained intensely with anti-Nantibodies (FIG. 1B).

We then proceeded with co-transfection of pUC57-S, pUC57-L andpUC57-Mv-eGFP(−) in FP-T7-infected BHK-21 cells. Whereas in previousexperiments using BSR-T7/5 cells, only a few eGFP-positive cells wereobserved, in these experiments, FACS analysis showed that 1.3% of thecells were positive for eGFP (FIG. 2). A similar experiment wasperformed where cells were co-transfected with pCAGGS-NSmGnGc. In thisexperiment, 2.5% of the cells were positive for eGFP (FIG. 2).Interestingly, the observation of clusters of eGFP-positive cellssuggested local spread of VLPs containing the reporter minigenome (datanot shown). After three days, the culture supernatant was collected andincubated with BHK-21 cells that were either untreated or transfectedwith the helper plasmids pCIneo-RVFV-L and pCAGGS-N. In cells that weretransfected with helperplasmids, 1.3% was positive for eGFP expression(FIG. 2). Moreover, 0.9% of the cells that were not previouslytransfected with helperplasmids was positive for eGFP expression,demonstrating that we were successful in producing replicon particlesthat contain all three genome segments (FIG. 2).

Production of RRPs Containing Two Genome Segments.

To facilitate further optimization of the system, we aimed to produceRRPs that contain only two genome segments. To this end, a reporterminigenome was produced in which the gene encoding the NSs gene of the Ssegment was exchanged for the gene encoding eGFP (i.e. S-eGFP). Previouswork demonstrated that the NSs gene is not essential for growth intissue culture (Muller et al. 1995. Am J Trop Med Hyg 53: 405-411) andother studies demonstrated that a virus containing this deletion isviable (Ikegami et al. 2006. J Virol 80: 2933-2940). Co-transfection ofthe resulting plasmid, pUC57-S-eGFP(−) with pUC57-L resulted inexpression of eGFP in a small percentage of cells. However, whenpCAGGS-NSmGnGc was added to the transfection mixture, 21.5% of the cellswere positive for eGFP, as determined by FACS analysis (FIG. 3).Incubation of BHK-21 cells with the collected supernatant resulted in4.7% of positive cells when helper plasmids were not provided, whereasthe number of cells increased to 28.7% when helper plasmids wereincluded (FIG. 3).

Production of Stable BHK-21 Cell Lines that Produce the Gn and GcGlycoproteins.

With the aim to produce a system for the continuous production of RRPs,stable cell lines were produced that constitutively produce the Gn andGc proteins. Briefly, BHK-21 cells were transfected with pCIneo-GnGc andclones with integrated plasmids were grown in the presence of geneticinG-418. A number of clones were tested for Gn/Gc expression by IPMA. Ahighly positive antiserum from a naturally infected sheep did not revealany Gn/Gc positive cells (data not shown). An antiserum derived from asheep that was previously vaccinated with NDV-GnGc was, however,successfully used to identify positive clones. One of the clones clearlydisplayed the highest Gn and Gc expression level as revealed by IPMA,although the expression levels appeared low (FIG. 4). This clone wastested for expression of Gc by Western blotting of proteins from celllysates. Using a previously described polyclonal antiserum specific fora Gc-derived peptide, Gc expression was clearly detected (FIG. 4C). Thiscell line was designated BHK-GnGc. To determine if VLPs were produced bythese cells, supernatants were ultracentrifuged and the proteins presentin the collected pellets were analyzed by Western blotting. The Gcprotein was not detected in the pellet fractions, suggesting that eitherno VLPs were produced, or that glycoprotein production was too low toallow detection.

Use of a cell line that maintains the RVFV L and S-eGFP segments isessential for the efficient production of RRPs.

Production of RRPs by infection of BHK-21 cells with FP-T7 andsubsequent introduction of plasmids providing the L segment, S-eGFPsegment and pCAGGS-NSmGnGc resulted in a maximum of RRP titers of 10E3to 10E4 TCID50/ml (data not shown). Although the BHK-GnGc cell lineappeared not suitable for the constitutive large scale production ofRRPs, it was striking to find that introduction of FP-T7 and subsequentintroduction of plasmids providing the L segment, S-eGFP segment andpCAGGS-NSmGnGc, resulted in clusters of positive cells that were largerin both number and size than those obtained with normal BHK-21 cells.

A cell line maintaining the RVFV L and S-eGFP segments could be highlyvaluable for several applications, including high-throughput screens ofantiviral agents outside biosafety containment facilities. We thereforeaimed to produce a cell culture of which each cell contains the RVFV Land S-eGFP segment. To this end, the FP-T7 virus was introduced,followed by introduction of the plasmids providing the L segment and theS-eGFP segment. To facilitate spread of the L and S-eGFP segment,pCAGGS-NSmGnGc was introduced several times after passage of the cells.Using this method, cell lines of both wildtype BHK-21 cells as well asBHK-GnGc were obtained of which most, if not all, cells expressed theeGFP reporter. However, whereas repetitive passage of theeGFP-expressing BHK-21 cells resulted in loss of eGFP, passage of theeGFP-expressing BHK-GnGc cells did not result in any loss of eGFPexpression. For at least 50 cell passages, eGFP expression in thesecells remained unchanged. When not transfected, BHK-GnGc cellscontaining the RVFV replicons were found to produce very small amountsof RRPs, with a maximum yield of 10E2 TCID50/ml. Importantly,introduction of pCAGGS-NSmGnGc or pCAGGS-GnGc in these cells resulted inRRP titers of maximally 10E6.8 or 10E6.4 TCID50/ml, respectively (FIG.5).

Efficient Production of RRPs Using NDV-GnGc.

Considering the superior production of GnGc by NDV when compared toexpression from plasmid, we tested if RRPs could be produced byinfection of the cells with NDV-GnGc. BHK-GnGc cells were co-infectedwith FP-T7 and NDV-GnGc and, after recovery, were transfected withpUC57-L and pUC57-S-eGFP(−). After 72 hrs of incubation, extremely large“comets” of positive cells were observed in the flasks (data not shown).This result suggested that large amounts of RRPs are produced whenNDV-GnGc is used to provide the glycoproteins. We subsequently split theculture into two flasks. One was left untreated, the other was againinfected with NDV-GnGc. The supernatants of these cells were collectedafter 48 h and the TCID50 RRP titers were determined on BHK-21 cells.The supernatant of the cells that were infected with NDV-GnGc twice,contained a titer of 10E7 TCID50/ml, whereas the supernatant of thecells that were infected only once contained a titer of 10E4.5TCID50/ml. The latter cell line was passaged 18 times, after which theRRP titer was again determined. Surprisingly, this revealed a titer of10E6 TCID50/ml. Moreover, visual examination of this cell line byfluorescence microscopy revealed that most, if not all, cells expressedeGFP (FIG. 6). This result led us to suggest that the cells werepersistently infected with NDFL-GnGc, thereby providing a continuoussource of Gn and Gc. Indeed, IPMAs using a mAb specific for the NDV Fprotein, revealed the presence of the virus in a subset of the cells. Itis important to note, however, that the virus present in the supernatantof these cells was shown to be non-infectious. This was expectedhowever, since lentogenic NDV strains such as the recombinant LaSotastrain used in the current work, requires cleavage of the F protein bytrypsin-like proteases for infectivity. In conclusion, by virtue of thepersistent infection of the cells with NDFL-GnGc, RRPs can continuouslybe produced to titers of up to 10E7 TCID50/ml.

To demonstrate that RRPs are incapable of autonomous spread, BHK cellswere infected with RRPs at a multiplicity of infection (m.o.i.) of 1.After two days, eGFP expression was observed by fluorescence microscopy(FIG. 8, left panel). BHK cells were incubated with collectedpre-cleared supernatant and after three days, cells were monitored foreGFP expression. No eGFP expression was observed, demonstrating that noprogeny infectious particles were produced by the RRP-infected BHK cells(FIG. 8, right panel). To establish the kinetics of RRP production,BHK-rep cells were transfected with pCAGGS-NSmGnGc and the culturemedium was collected at different time points post transfection. RRPswere titrated on BHK cells using eGFP expression as the readoutparameter. This experiment demonstrated that a titer close to 106TCID50/ml was obtained already after 22 h (FIG. 9).

To visualize RRP proteins, RRPs were pelleted by ultracentrifugation.The proteins were separated in NuPAGE gels, transferred tonitrocellulose membranes and detected using peptide antisera specificfor the Gn and Gc protein or a monoclonal antibody specific for the Nprotein. Analysis of the supernatant obtained from non-transfectedBHK-Rep cells revealed only the N protein (FIG. 10). Interestingly, thisresult suggests that the RVFV N protein is released from cells,presumably in the form of ribonucleoprotein core particles, resemblingresults previously described from studies on CCHFV (Bergeron et al.,2007. J Virol 81: 13271-13276). Analysis of supernatant from BHK-Repcells transfected with pCAGGS-NSmGnGc (pCAGGS-M) revealed the Gnprotein, the NSm protein, the Gc protein and the N protein (FIG. 10).

Example 2 Production of Bunyavirus Replicon Particles (BRPs) ofCrimean-Congo Hemorrhagic Fever Virus Strain IbAr10200

cDNA encoding anti virus-sense full-length RNA of the L (GenBank:AY389508.2), M (GenBank: AF467768.2) and S (GenBank: U88410.1) genomesegments CCHFV strain IbAr10200 are synthesized and flanked by a T7polymerase promoter and cDNA encoding a HDV ribozyme sequence. A T7transcription termination sequence is preferably positioned downstreamof the ribozyme cDNA. These sequences are cloned into pUC57 vectorsessentially as exemplified in Example 1, resulting in pUC57-CCHFV-L,pUC57-CCHFV-M and pUC57-CCHFV-S.

To facilitate cloning into pUC57 of the cDNA encoding the CCHFV L genomesegment, a KpnI restriction site is introduced immediately upstream ofthe T7 promoter sequence and immediately downstream of the T7transcription termination sequence.

To facilitate cloning of the cDNA encoding the CCHFV M genome segment, aKpnI restriction site is introduced immediately upstream of the T7promoter sequence and a SalI restriction site is introduced immediatelydownstream of the T7 transcription termination sequence.

To facilitate cloning of the cDNA encoding the CCHFV S genome segment,an EcoRI restriction site is introduced immediately upstream of the T7promoter sequence and a BamHI restriction site is introduced immediatelydownstream of the T7 transcription termination sequence.

The complete open reading frame of the M genome segment (M-ORF,nucleotides 93-5147 of GenBank sequence AF467768.2) is introduceddownstream of the CMV promoter of pCIneo. Stable cell lines are producedby transfecting pCIneo-M-ORF into BHK-21 cells and cells with integratedplasmids are selected by culturing in the presence of G-418.

As an alternative, BHK-21 cells are provided with the CCHFV M-ORFencoded proteins by infecting the eukaryotic cell with a recombinantviral vector that transduces the CCHFV M-ORF-encoded polyprotein,followed by selecting a cell in which the recombinant viral vector ispersistently present without causing overt cytopathogenic effect.

The cells that (conditionally or constitutively) express the CCHFVM-ORF-encoded proteins are infected with FP-T7 and, after recovery,transfected with plasmids pUC57-CCHFV-L and pUC57-CCHFV-S. When requiredfor efficient production, a construct encoding the CCHFV structuralglycoproteins under the control of a suitable Polymerase-II promoter(such as the CAG promoter in the pCAGGS plasmid) is also transfected orinfected to provide the CCHFV structural glycoproteins. This procedureresults in the production of CCHFV replicon particles that do notcontain a CCHFV M genome segment.

The T7 promoter that is used has the nucleotide sequence:5′-TAATACGACTCACTATAG-3′

The HDV ribozyme sequence that is used has the nucleotide sequence5′-GGGTCGGCATGGCATCTCC-3′.

The T7 terminator sequence that is used has the nucleotide sequence:5-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG-3′

Example 3

Production of bunyavirus replicon particles (BRPs) of Dobrava-Belgradevirus (DOBV) strain DOBV/Ano-Poroia/Afl9/1999

cDNA encoding anti virus-sense full-length RNA of the L (GenBank:AJ410617.1), M (GenBank: AJ410616.1) and S (GenBank: AJ410615.1) genomesegments DOBV strain DOBV/Ano-Poroia/Afl9/1999 is synthesized andflanked by a T7 polymerase promoter and cDNA encoding a HDV ribozymesequence. A T7 transcription termination sequence is preferably presentdownstream of the introduced ribozyme cDNA. These sequences are clonedinto pUC57 vectors essentially as exemplified in Example 1, resulting inpUC57-DOBV-L, pUC57-DOBV-M and pUC57-DOBV-S.

To facilitate cloning into pUC57 of the cDNA encoding the DOBV L genomesegment, a BamHI restriction site is introduced immediately upstream ofthe T7 promoter sequence and immediately downstream of the T7transcription termination sequence.

To facilitate cloning of the cDNA encoding the DOBV M genome segment, aKpnI restriction site is introduced immediately upstream of the T7promoter sequence and a SalI restriction site is introduced immediatelydownstream of the T7 transcription termination sequence.

To facilitate cloning of the cDNA encoding the DOBV S genome segment, aKpnI restriction site is introduced immediately upstream of the T7promoter sequence and a SalI restriction site is introduced immediatelydownstream of the T7 transcription termination sequence.

The complete open reading frame of the M genome segment (MDOBV-ORF,nucleotides 41-3448 of GenBank sequence AJ410616.1) is introduceddownstream of the CMV promoter of pCIneo. Stable cell lines are producedby transfecting pCIneo-MDOBV-ORF into BHK-21 cells and selecting cellswith integrated plasmids by culturing in the presence of G-418.

Alternatively, the eukaryotic cell is provided with MDOBV-ORF-encodedproteins by infecting the eukaryotic cell with a recombinant viralvector that produces the MDOBV proteins, followed by selecting a cell inwhich the recombinant viral vector is persistently present withoutcausing overt cytopathogenic effect.

The cells that (conditionally or constitutively) express theMDOBV-ORF-encoded proteins are infected with FP-T7 and, after recovery,transfected with plasmids pUC57-DOBV-L and pUC57-DOBV-S. When requiredfor efficient production, a construct encoding the DOBV structuralglycoproteins under the control of a suitable Polymerase-II promoter(such as the CAG promoter in the pCAGGS plasmid) is also transfected orinfected to provide the DOBV structural glycoproteins. This procedureresults in the production of DOBV replicon particles.

The T7 promoter that is used has the nucleotide sequence:5′-TAATACGACTCACTATAG-3′

The HDV ribozyme sequence that is used has the nucleotide sequence5′-GGGTCGGCATGGCATCTCC-3′.

The T7 terminator sequence that is used has the nucleotide sequence:5-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG-3′

Example 4

Tet-Off system for the inducible expression of RVFV NSm, Gn and Gcproteins

BHK-21 cells are transfected with the pTet-Off Advanced plasmid(Clontech, CA, USA) according to the instructions of the manufacturers.After selection with G-418, resistant clones are selected, yielding aBHK-Tet-Off Advanced cell line.

The complete open reading frames of the M segment of a RVFV strain35/74, (nucleotides 21-3614) are synthesized with flanked KpnI (5′ end)and NotI and SalI (3′ end) restriction sites and cloned into pUC57 usingKpnI and SalI restriction sites (GenScript Corporation). The inserts arereleased from the pUC57 plasmids by KpnI/NotI digestion and cloned intothe KpnI/NotI-digested pTREtight vector (Clontech).

The BHK-Tet-Off Advanced cells are transfected with pTREtight-NSmGnGcand a linear marker that facilitates the selection of transfected cellsby hygromycin or puromycin. Clones that produce the proteins of interestare selected by growing the clones in the absence of doxycycline (DOX).

After selection of suitable clones, the cells are grown in the presenceof DOX and infected with FP-T7. After recovery, the cells aretransfected with pUC57-L and pUC57-S-eGFP. After recovery, the culturemedium of the cells is replaced by medium without DOX, resulting inexpression of NSm, Gn and Gc and the formation of RVFV repliconparticles.

Example 5 RVFV Replicon Particles that Produce Recombinant SolubleMultimeric HA of Pandemic Swine-Origin 2009 A (H1N1) Influenza Virus

Oligo MSC-1 and Oligo MSC-2 are synthesized. Annealing of Oligo MCS-1and Oligo MCS-2 results in a double-stranded DNA molecule containingNcoI and XbaI overhangs and additional SpeI, XhoI, BglI, NotIrestriction sites.

Oligo MCS 1: 5′-CATGGACTAGTCTCGAGGCTAGCAGATCTGCGGCCGCT-3′ Oligo MCS 2:5′-CTAGAGCGGCCGCAGATCTGCTAGCCTCGAGACTAGTC-3′

The eGFP gene is removed from pUC57-S-eGFP (FIG. 7F) by NcoI/XbaIdigestion and the MCS linker is ligated into this vector, yieldingpUC57-S-MCS.

The sequence listed below (Seq CD5-HA-GCN4-ST) encodes a humancodon-optimized soluble hemagglutinin ectodomain (sHA, amino acids 17 to522) of influenza virus A/California/04/2009 (H1N1). This sequence issynthesized at the GenScript Corporation. The HA gene is preceded by asequence encoding an N-terminal CD5 signal peptide and followed bysequences encoding a C-terminal artificial GCN4 trimerization domain(GCN4-pII, Harbury et al. 1993. Science 262: 1401-1407) and aStreptavidin-tag (Strep) for affinity purification. This construct isdescribed in Bosch et al. 2010. J. Virol. 84: 10366-10374).

To generate a vector comprising CD5-sHA-GCN4-pII, pUC57-S-MCS isdigested with NheI/XbaI and NheI/XbaI digested HA-GCN4-ST sequence iscloned into this plasmid, yielding pUC57-S-HA-GCN4-ST. The CD5 sequenceis subsequently introduced by annealing of the following oligo's:

Oligo CD5-1: 5′- CTAGTATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTG-3′ Oligo CD5-2: 5′-CTAGCACGGAAGCGACCAGCATCCCCAGCAGGTACAAGGTGGCCAGCGGTTGCAGAGACCCCATGGGCATA-3′

The CD5 linker is ligated into SpeI/NheI-digested pUC57-S-HA-GCN4-ST,yielding pUC57-S-CD5-HA-GCN4-ST.

BHK-GnGc cells are infected with FP-T7. After recovery, the cells aretransfected with pUC57-L, pUC57-S-CD5-HA-GCN4-ST and pCAGGS-NSmGnGc,essentially as exemplified in Example 1. After cell passage, thetransfection is repeated. This sequence of events is repeated until allthe cells of the monolayer express the CD5-HA-GCN4-ST protein, as isdetermined by IPMA. The quantity of HA-GCN4-ST in the culture medium ofthese cells is determined. To establish high levels of NSmGnGc proteins,plasmid pCAGGS-NSmGnGc is again introduced in the cell line, yieldingreplicon particles containing the S-CD5-HA-GCN4-ST gene. The repliconparticles are used for vaccination/challenge experiments in mice toestablish the protective efficacy against a lethal H1N1 challenge.

Seq. CD5-HA-GCN4-ST

Nucleotides 1-69 (underlined) encode the CD5 sequence

Nucleotides 70-1590 encode the HA ectodomain

Nucleotides 1591-1713 (underlined) encode the GCN4 domain

Nucleotides 1714-1737 encode the Strep tag

atgcccatggggtctctgcaaccgctggccaccttgtacctgctggggatgctggtcgct M  P  M  G  S  L  Q  P  L  A  T  L  Y  L  L  G  M  L  V  A  tccgtgctagcagacaccctgtgcatcggctaccacgccaacaacagcaccgacaccgtg S  V  L  A  D  T  L  C  I  G  Y  H  A  N  N  S  T  D  T  Vgacaccgtgctggagaagaacgtgaccgtgacccacagcgtgaacctgctggaggacaag D  T  V  L  E  K  N  V  T  V  T  H  S  V  N  L  L  E  D  Kcacaacggcaagctgtgcaagctgcgcggcgtggctccactgcacctgggcaagtgcaac H  N  G  K  L  C  K  L  R  G  V  A  P  L  H  L  G  K  C  Nattgctggatggatcctgggaaacccagagtgcgagagcctgagcaccgccagcagctgg I  A  G  W  I  L  G  N  P  E  C  E  S  L  S  T  A  S  S  Wagctacatcgtggagacccccagcagcgacaacggcacctgctaccccggcgacttcatc S  Y  I  V  E  T  P  S  S  D  N  G  T  C  Y  P  G  D  F  Igactacgaggagctgcgcgagcagctgagcagcgtgagcagcttcgagcgcttcgagatc D  Y  E  E  L  R  E  Q  L  S  S  V  S  S  F  E  R  F  E  Ittccccaagaccagcagctggccaaaccacgacagcaacaagggagtgaccgctgcttgc F  P  K  T  S  S  W  P  N  H  D  S  N  K  G  V  T  A  A  Cccacacgctggagccaagagcttctacaagaacctgatctggctggtgaagaagggcaac P  H  A  G  A  K  S  F  Y  K  N  L  I  W  L  V  K  K  G  Nagctaccccaagctgagcaagagctacatcaacgacaagggcaaggaggtgctggtgctg S  Y  P  K  L  S  K  S  Y  I  N  D  K  G  K  E  V  L  V  Ltggggcatccaccaccccagcaccagcgccgaccagcagagcctgtaccagaacgccgac W  G  I  H  H  P  S  T  S  A  D  Q  Q  S  L  Y  Q  N  A  Dacctacgtgttcgtgggcagcagccgctacagcaagaagttcaagcccgagatcgccatc T  Y  V  F  V  G  S  S  R  Y  S  K  K  F  K  P  E  I  A  Icgccccaaggtgcgcgaccaggagggccgcatgaactactactggaccctggtggagccc R  P  K  V  R  D  Q  E  G  R  M  N  Y  Y  W  T  L  V  E  Pggcgacaagatcacctttgaggctaccggaaacctggtggtgccacgctacgcttttgct G  D  K  I  T  F  E  A  T  G  N  L  V  V  P  R  Y  A  F  Aatggagaggaatgctggcagcggcatcatcatcagcgacacccccgtgcacgactgcaac M  E  R  N  A  G  S  G  I  I  I  S  D  T  P  V  H  D  C  Naccacctgccagacccccaagggcgccatcaacaccagcctgcccttccagaacatccac T  T  C  Q  T  P  K  G  A  I  N  T  S  L  P  F  Q  N  I  Hcccatcaccatcggcaagtgccccaagtacgtgaagagcaccaagctgcgcctggccacc P  I  T  I  G  K  C  P  K  Y  V  K  S  T  K  L  R  L  A  Tggactgaggaacatcccaagcatccagagccgcggcctgtttggagctattgctggattc G  L  R  N  I  P  S  I  Q  S  R  G  L  F  G  A  I  A  G  Fattgagggcggctggaccggaatggtggatggatggtacggctaccaccaccagaacgag I  E  G  G  W  T  G  M  V  D  G  W  Y  G  Y  H  H  Q  N  Ecagggcagcggctacgccgccgacctgaagagcacccagaacgccatcgacgagatcacc Q  G  S  G  Y  A  A  D  L  K  S  T  Q  N  A  I  D  E  I  Taacaaggtgaacagcgtgatcgagaagatgaacacccagttcaccgccgtgggcaaggag N  K  V  N  S  V  I  E  K  M  N  T  Q  F  T  A  V  G  K  Ettcaaccacctggagaagcgcatcgagaacctgaacaagaaggtggacgacggcttcctg F  N  H  L  E  K  R  I  E  N  L  N  K  K  V  D  D  G  F  Lgacatctggacctacaacgccgagctgctggtgctgctggagaacgagcgcaccctggac D  I  W  T  Y  N  A  E  L  L  V  L  L  E  N  E  R  T  L  Dtaccacgacagcaacgtgaagaacctgtacgagaaggtgcgcagccagctgaagaacaac Y  H  D  S  N  V  K  N  L  Y  E  K  V  R  S  Q  L  K  N  Ngccaaggagatcggcaacggctgcttcgagttctaccacaagtgcgacaacacctgcatg A  K  E  I  G  N  G  C  F  E  F  Y  H  K  C  D  N  T  C  Mgagagcgtgaagaacggcacctacgactaccccaagtacagcgaggaggccaagctgaac E  S  V  K  N  G  T  Y  D  Y  P  K  Y  S  E  E  A  K  L  NCgcgaggagatcgacggcgtgaagctcgagttaattaagcgcatgaagcagatcgaggac R  E  E  I  D  G  V  K  L  E  L  I  K  R  M  K  Q  I  E  D aagatcgaagagatcgagtccaagcagaagaagatcgagaacgagatcgcccgcatcaag K  I  E  E  I  E  S  K  Q  K  K  I  E  N  E  I  A  R  I  K Aagattaagctggtgccgcgcggcagcctcgagtggagccacccgcagttcgagaagtga K  I  K  L  V  P  R  G  S  L  E  W  S  H  P  Q  F  E  K  -

Example 6 RRP Infection of Mammalian and Insect Cells and Production ofRRPs

To determine if other mammalian and insect cells can be infected withRRPs, Human Embryonic Kidney 293 cells (HEK293T), Drosophila S2 cellsand Aedes albopictus C6/36 cells were infected with RRPs at an m.o.i.of 1. Of note, the m.o.i. was calculated using the titer determined onBHK cells. This experiment demonstrated that both mammalian and insectcells can be readily infected with RRPs, although reporter geneexpression in insect cells is considerably lower (FIG. 11A). Expressionof eGFP in mammalian cells and insect cells was optimal at 42 or 72hours post infection (hpi), respectively (FIG. 11).

Next, we were interested in determining if HEK293T cells can be used forthe production of RRPs. To this end, BHK cells and HEK293T cells wereinfected with RRPs at an m.o.i. of 3. After three days, the cells wereseeded in a 6-well plate and transfected with pCAGGS-NSmGnGc. Thesupernatant was collected after three days and the RRP titers obtainedwere 10E7 (10⁷) or BHK cells and 10E6.5 (10^(6.5)) TCID50/ml for HEK293Tcells. This result demonstrates that wildtype BHK cells and HEK293Tcells can be used for the production of RRPs by combining an RRPinfection with a transfection of the pCAGGS-NSmGnGc plasmid

Example 7 Use of RRPs in a Virus Neutralization Test (VNT)

Classical VNT and a novel VNT that uses RRPs instead of live virus(RaPid VNT) were performed with sera from lambs that were previouslyexperimentally infected with the 35/74 virus. To confirm the presence ofRVFV-specific antibodies, the sera were analyzed by the recN RVFV ELISA(BDSL, Ayrshire Scotland, UK) prior to analysis by VNT. The classicalVNT was performed as described previously (de Boer et al., 2010. Vaccine28: 2330-2339). For the RaPid VNT, serum dilutions were prepared in96-well plates in 50 μl GMEM supplemented with 5% FBS, 4% TPB, 1% MEMNEAA, 1% pen/strep. Growth medium containing ˜200 RRPs in a 50 μl volumewas added to the serum dilutions and incubated for 1.5 h at roomtemperature. Next, 50 μl of growth medium containing 40 000 BHK cellswas added to each well. Plates were incubated at 37° C. and 5% CO2.After 36-48 hrs the neutralization titer was calculated using theSpearman-Kärber method (Karber (1931). Arch Exp Path Pharmak162:480-483; Spearman (1908). Br J Psychol 2: 227-242).

TABLE 1 Comparison of the classical VNT assay with the RaPid VNT. Serafrom experimentally infected lambs were analysed by the recN ELISA(ELISA), classical VNT, and RaPid VNT. Neutralization titres aredetermined as ¹⁰log 50% end-point titres. Lamb Classical no: VNT RaPidVNT ELISA 4308 3.56 3.94 POS 4309 4.09 4.16 POS 4310 0 0 NEG 4311 4.014.24 POS 4312 3.71 4.76 POS 4314 3.56 4.46 POS 4315 3.71 4.39 POS 43184.16 4.39 POS 4321 0 0 NEG 4324 4.24 4.31 POS 4328 4.01 4.69 POS

This experiment revealed that the use of RRPs in the so-called RaPid VNThas an optimal readout between 36 and 48 hrs and is of equal, if nothigher sensitivity than the classical VNT (Table 1).

Example 8 Vaccination and Challenge of Mice

Female BALB/c mice (Charles River laboratories, Maastricht, TheNetherlands) were housed in groups of five animals in type-IIIfilter-top cages and kept under biosafety level-3 containment. Groups of10 mice were vaccinated via the intramuscular or subcutaneous routeeither once on day 21 or two times on days 0 and 21 with 10E6 TCID50 ofRRPs in 50 μl PBS. One group of nine mice was left untreated(non-vaccinated). The body weights of the mice were monitored weekly. Onday 42, all mice were challenged via the intraperitoneal route with10E2.7 TCID50 of RVFV strain 35/74 in 0.5 ml culture medium. Challengedmice were monitored daily for visual signs of illness and mortality.This experiment was approved by the Ethics Committee for AnimalExperiments of the Central Veterinary Institute of Wageningen Universityand Research Centre.

To study the vaccine efficacy of RRPs, groups of 10 mice were immunizedwith 50 μl of an inoculum containing 10E6 TCID50 RRPs, via either thesubcutaneous or intramuscular route, either once or twice, with a threeweek interval. One group of 9 non-vaccinated mice was added as a controlgroup. The mice were challenged on day 42 with a known lethal dose ofRVFV strain 35/74. All non-vaccinated mice displayed overt clinicalsigns and weight loss and eight of a total of nine non-vaccinated micesuccumbed to the infection within four days after challenge. One mousesurvived for twelve days, but eventually died. The percentage ofsurvival in the groups of mice vaccinated either once or twice via thesubcutaneous route was 60% (FIG. 12). In contrast, 100% of the micevaccinated via the intramuscular route, either once or twice, survivedthe challenge (FIG. 12). These mice did not show any clinical signs orweight loss throughout the experiment. This demonstrates that a singleintramuscular vaccination with 10E6 RRPs can protect mice from a lethaldose of RVFV.

Example 9 Production of Bunyavirus Replicon Particles of Severe Feverwith Thrombocytopenia Syndrome (SFTS) Virus Isolate HB29

SFTS virus was first described by Yu et al. (Yu X J, et al. Fever withThrombocytopenia Associated with a Novel Bunyavirus in China. N Engl JMed 2011 Mar. 16).

cDNAs encoding full-length viral RNA corresponding to the L (GenBank:HM745930.1), M (GenBank: HM745931.1) and S (GenBank: HM745932.1) genomesegments of SFTS virus isolate HB29 are synthesized and flanked by a T7polymerase promoter and cDNA encoding a HDV ribozyme sequence. A T7transcription termination sequence is preferably positioned downstreamof the ribozyme cDNA. These sequences are cloned into pUC57 vectorsessentially as exemplified in Example 1, resulting in pUC57-SFTS-L,pUC57-SFTS-M and pUC57-SFTS-S.

To facilitate cloning into pUC57 of the cDNA encoding the SFTS L genomesegment, a KpnI restriction site is introduced immediately upstream ofthe T7 promoter sequence and immediately downstream of the T7transcription termination sequence.

To facilitate cloning of the cDNA encoding the SFTS M genome segment, aKpnI restriction site is introduced immediately upstream of the T7promoter and immediately downstream of the T7 transcription terminationsequence.

To facilitate cloning of the cDNA encoding the SFTS S genome segment, aKpnI restriction site is introduced immediately upstream of the T7promoter sequence and immediately downstream of the T7 transcriptiontermination sequence.

An additional construct encoding an SFTS S genome-like segment in whichthe NSs gene (nucleotides 835-1716 of GenBank: HM745932.1) is replacedby a gene encoding a suitable reporter protein (i.e. eGFP or luciferase)is also developed. A plasmid encoding an S-like genome segment in whichthe NSs gene is replaced by the eGFP gene is named pUC57-SFTS-S-eGFP.Similar as was shown for RVFV, introduction of the SFTS-L andSFTS-S-eGFP genome segments in suitable cells (e.g. BHK-21 cells)results in viable cells maintaining these genome segments.

The complete open reading frame of the M genome segment (M-ORF,nucleotides 19-3240 of GenBank sequence HM745931.1) is introduceddownstream of the CMV promoter of pCIneo. Stable cell lines are producedby transfecting pCIneo-SFTS-M-ORF into BHK-21 cells and cells withintegrated plasmids are selected by culturing in the presence of G-418.

As an alternative, BHK-21 cells are provided with the SFTS-M-ORF encodedproteins by infecting the eukaryotic cell with a recombinant viralvector that transduces the SFTS M-ORF-encoded polyprotein and a cellline is selected in which the recombinant viral vector is persistentlypresent without causing overt cytopathogenic effect.

The cells expressing the SFTS M-ORF-encoded proteins are infected withFP-T7 and, after recovery, transfected with plasmids pUC57-SFTS-L andeither pUC57-SFTS-S or pUC57-SFTS-S-eGFP. When required for efficientproduction, a construct encoding the SFTS structural glycoproteins underthe control of a suitable Polymerase-II promoter (such as the CAGpromoter in the pCAGGS plasmid) is also transfected or infected toprovide the SFTS structural glycoproteins. This procedure results in theproduction of SFTS replicon particles of isolate HB29 that do notcontain an SFTS M genome segment.

The T7 promoter that is used has the nucleotide sequence:5′-TAATACGACTCACTATAG-3′

The HDV ribozyme sequence that is used has the nucleotide sequence5′-GGGTCGGCATGGCATCTCC-3′.

The T7 terminator sequence that is used has the nucleotide sequence:5-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG-3′

A similar method can be used to produce replicon particles of other SFTSisolates.

Example 10 Rapid Immunity Against RVFV in Sheep after a SingleVaccination with the NSR Vaccine

Materials and Methods

Preparation of the Challenge Virus

The RVFV virus that was used for challenge was rescued from cDNA (seeExample 1 and Kortekaas et al. 2011. J. Virol. Accepted forpublication). The recombinant 35/74 (rec35/74) virus was titrated onBHK-21 cells and the titer was determined as tissue culture infectivedose 50 (TCID50) using the Spearman-Kärber algorithm (Example 1). Thecomplete sequence of the L, M and S genome sequences of the 35/74isolate can be found on GenBank under the accession numbers JF784386,JF784387 and JF784388, respectively.

2.2 Preparation of the Vaccines

The ectodomain of the Gn protein (GneS3) was produced from the insectexpression vector pMT/BiP/V5-HisA (Invitrogen, Carlsbad, Calif., USA).The sequence encoding the authentic Gn signal peptide was replaced by asequence coding for the BiP signal peptide, specifying the junctionsequence “GLSLG-RSLRSLAEDPH”, in which GLSLG denotes the BiP, RSLRSLdenotes a linker sequence, and AEDPH the start of the Gn ectodomain. Inthe pMT-GneS3 plasmid, the Gn ectodomain sequence was fused to asequence encoding a combined FLAG-tag/enterokinase (EK) cleavage sitefor easy detection and purification of the monomeric protein (DYKDDDDK)and three Strep tags (WSHPQFEK) separated by glycine linkers(GGGSGGGSGGGS), resulting in the following sequence: ( . . .YQCHTDPTGDYKDDDDKAGPGWSHPQFEK GGGSGGGSGGGSWSHPQFEKGGGSGGGSGGGSWSHPQFEKin which the sequences resulting from introduced restriction sites areindicated in bold. The enterokinase cleavage site was introduced toallow removal of the Strep-tag after purification.

The secreted Gn ectodomain was purified from the cell culturesupernatant by virtue of its C-terminal 3× Strep-tag using Strep-TactinSepharose according to the manufacturer's recommendations (IBA,Göttingen, Germany). The GneS3 protein was eluted from the washed beadswith 4 mM D-Desthiobiotin (IBA) and concentrated using an Amicon®Ultra-4 concentrator with a molecular mass cut-off of 30 kDa (Millipore,Billerica, Mass., USA). The protein, named GneS3, was formulated inStimune water-in-oil adjuvant (Prionics, Lelystad, The Netherlands) to afinal concentration of 20 μg/ml.

Production of the NDFL-GnGc was previously reported (Kortekaas et al.2010. Vaccine 28: 4394-4401). OBP vaccine is a commercially availableinactivated RVFV vaccine (Onderstepoort Biological Products [OBP],Onderstepoort, South Africa). This vaccine was purchased from OBP andadministered according to the instructions of the manufacturer. Theadministered doses and routes of vaccination of the four indicatedvaccines are depicted in Table 2.

TABLE 2 Route and dose of vaccines Vaccine Route Dose Adjuvant OBPvaccine Subcutaneous According to Aluminium protocol of hydroxide gelmanufacturer NDFL-GnGc Intramuscular 2.10⁷ TCID₅₀ None NSR Intramuscular10⁷ TCID₅₀ None GneS3 Subcutaneous 20 μg Stimune water-in-oil

2.3 Vaccination and Challenge

Thirty conventional European breed lambs were purchased from acommercial sheep farm in The Netherlands and divided over five groups.Lambs were vaccinated once at the age of six weeks (day 0), as depictedin Table 2. On day 19 (days post challenge [DPC] 0), all lambs werechallenged via the intraperitoneal route with 10⁵ TCID50 of RVFVrec35/74. EDTA blood samples were collected daily starting from day 19(DPC 0) until day 26 (DPC 7) and again on days 28, 30, 33, 35, 37 and 40(DPC 9, 11, 14, 15, 17 and 20). Serum samples were collected on days −7,0, 7, 14, and daily from day 19 (day of challenge) to 26 (DPC 7) andfinally on days 33 (DPC 14) and 40 (DPC 20). Body weights weredetermined weekly, on DPI −7, −1, 6, 13, 18, 25, 32 and 39. Rectal bodytemperatures were determined daily starting on day 17 (two days prior tochallenge) until the end of the experiment.

2.4 Quantitative Real-Time PCR and ELISA

Viral RNA was isolated from plasma samples using the QuickGene DNAtissue kit S (DT-S, Fuji Photo Film Europe GmbH, Dusseldorf, Germany)with the following modifications. Proteinase K solution (EDT, DT-S kit,30 μl) and 3 μl polyadenylic acid A (polyA 5 μg/μl, Sigma, St. Louis,Mo., USA) were added to 250 μl lysis buffer (LDT, DT-S kit). Of thismixture, 250 μl was added to 300 μl plasma. The mixture was heated at72° C. for 10 min in a heating block and stored at −20° C. until furtheranalysis. RNA isolation was subsequently performed using the QG-Mini80Workflow (Fuji Film). The lysate was mixed with 350 μl 99% ethanolbefore loading on the column. After three wash steps with 750 μl washbuffer (WDT, DT-S kit) the RNA was eluted with 50 μl elution buffer(CDT, DT-S kit). The material was stored at −70° C. until furtheranalysis.

RNA samples (5 μl) were used for quantitative Taqmanreverse-transcriptase real-time PCR (qRRT-PCR). The LightCycler RNAAmplification Kit HybProbe (Roche, Almere, The Netherlands) was used andprimers, probes and cycling conditions were used as previously described(Drosten et al. 2002 J Clin Microbiol 40: 2323-2330).

2.5 Clinical Chemistry

Clinical chemistry was performed with serum collected on the day ofchallenge (study day 19, DPC 0) and subsequently on days 20-25 (DPC1-6), and on days 32 (DPC 14) and 38 (DPC 21). Enzyme analysis wasperformed using the Spotchem EZ SP-4430 analyser (Menarini Diagnostics,Valkenswaard, The Netherlands) using strips capable of detectingalkaline phosphatase (ALP), alanine transaminase (ALT), creatinine,glucose, total protein and urea.

On day 0, blood samples (n=30) were collected and used to define theupper and lower limits of blood parameters. Limits were set at theaverage upper and lower averages plus two times their standarddeviations.

2.6 Statistical Analysis

Statistical analyses were performed with the one-way analysis ofvariance (ANOVA) with Bonferroni's multiple comparison test usingGraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego,Calif., USA). Statistical differences with p-values<0.05 were consideredsignificant.

Results

After acclimatization for one week, all lambs were vaccinated asdepicted in Table 2. On different time points after vaccination, theinjection sites were inspected for possible adverse reactions. Theseinspections revealed mild to moderate swelling in four and five out ofsix lambs vaccinated with the OBP and GneS3 vaccine, respectively. Inthe other groups, no injection site reactions were observed (FIG. 13).No adverse reactions at the injection site were observed in lambsvaccinated with the NDV-GnGc vaccine or the NSR vaccine.

After challenge the rectal temperatures in the control group peaked at 2DPC (FIG. 14). Peak rectal temperatures and the total days of fever(rectal body temperature ≧40.1° C.) were both significantly lower(p<0.0001) in all vaccinated groups, compared to the control group.

The occurrence of viremia was determined by qRRT-PCR on RNA isolatedfrom plasma samples. High levels of viral RNA were detected in all butone of the unvaccinated control animals, peaking on DPC 3 (FIG. 15).Viral RNA levels were significantly lower in all vaccinated groups(p<0.0001).

The body weights of the lambs were determined weekly. The lambs in theunvaccinated control group all displayed weight loss in the first twoweeks after challenge (between days 18-25). Some weight loss, at a latertime point (between days 25-32), was noted in five lambs vaccinated withNDV-GnGc and two lambs vaccinated with the NSR vaccine (FIG. 16).Differences between body weights were not statistically significant.

To investigate the occurrence of liver and renal damage, biochemicalanalyses were performed on serum samples using the Spotchem EZ drychemistry analyzer. Hepatic dysfunction was assessed by sequentialmeasurements of serum alkaline phosphatase (ALP), alanine transaminase(ALT) and total protein concentrations (TP) (FIG. 17). Total proteinconcentration is assumed to represent mostly albumin levels. Inunvaccinated lambs, ALP and ALT levels were clearly increased whencompared to vaccinated lambs. Statistical significance (p<0.05) washowever only achieved when comparing ALP levels in plasma obtained fromunvaccinated lambs (Mock) and NDV-GnGc-vaccinated lambs.

The concentrations of blood urea nitrogen (BUN) and creatinine weremeasured to assess renal function. BUN levels in serum obtained fromunvaccinated lambs were clearly on average higher than levels detectedin the serum of vaccinated lambs (FIG. 17).

Creatinine levels in serum from unvaccinated lambs were significantlyhigher when compared to levels detected in serum from vaccinated lambs(OBP vaccine, p<0.0005; GneS3, p<0.005; NSR, p<0.005; NDV-GnGc, p<0.05).

One lamb in the Mock-vaccinated control group succumbed to theinfection. In this lamb, creatinine levels peaked on DPC 8 to a level of582 μmol/l and BUN levels peaked to 30.6 mmol/l. This strongly suggeststhat this lamb died from severe kidney failure. Liver failure wasrevealed by a peak in ALT concentration of 20 U/l.

Conclusions

We here demonstrate that a single vaccination of sheep with thenon-spreading NSR vaccine prevents mortality and morbidity andsignificantly reduces viremia, fever and clinical signs resulting from aRVFV challenge. The efficacy of the NSR vaccine did not significantlydiffer from the other vaccines evaluated in our study. The highimmunogenicity of the NSR vaccine explains that the use of adjuvants isnot required. The inability of the NSR vaccine to spread from thevaccinated animal provides optimal safety.

Example 11 RVFV Replicon Particles that Produce Recombinant SolubleMultimeric NA of Pandemic Swine-Origin 2009 A (H1N1) Influenza Virus

Seq S-CD5-OS-GCN4-NA (see below) encodes a human codon-optimized solubleneuraminidase ectodomain (NA amino acids 75 to 469) of influenza virusA/California/04/2009 (H1N1). This sequence was synthesized at theGenScript Corporation. The NA gene is preceded by a sequence encoding anN-terminal CD5 signal peptide which is followed by sequences encoding aN-terminal OneSTrEP (OS) comprising a purification motif and atetramerization motif (GCN4-pLI; Bosch et al. 2010. J. Virol. 84:10366-10374).

To generate a vector comprising CD5-OS-GCN4-NA, pUC57-S-MCS was digestedwith NheI and XbaI and NheI/XbaI digested OS-GCN4-NA sequence was clonedinto this plasmid, yielding pUC57-S-GCN4-NA. The CD5 sequence wassubsequently introduced by annealing of the following oligo's:

Oligo CD5-3: 5′- CATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTG-3′ Oligo CD5-4: 5′-CTAGCACGGAAGCGACCAGCATCCCCAGCAGGTACAAGGTGGCCAGCGGT TGCAGAGACCCCATGGG-3′

The CD5 linker is ligated into NcoI/NheI-digested pUC57-S-GCN4-NA,yielding pUC57-S-CD5-OS-GCN4-NA.

BHK-GnGc cells were infected with FP-T7. After recovery, the cells weretransfected with pUC57-L, pUC57-S-CD5-OS-GCN4-NA and pCAGGS-NSmGnGc,essentially as exemplified in Example 1. After cell passage, thetransfection was repeated. This sequence of events was repeated untilall the cells of the monolayer express the CD5-OS-GCN4-NA protein, asdetermined by IPMA. The quantity of GCN4-NA in the culture medium ofthese cells was determined. To establish high levels of NSmGnGcproteins, plasmid pCAGGS-NSmGnGc was again introduced in the cell line,yielding replicon particles containing the S-CD5-OS-GCN4-NA gene. Thereplicon particles are used for vaccination/challenge experiments inmice to establish the protective efficacy against a lethal H1N1challenge.

Seq. S-CD5-OS-GCN4-NA

Nucleotides 1-69 (underlined) encode the CD5 sequence

Nucleotides 73-156 encodes the OneSTrEP(OS) peptide

Nucleotides 163-261 encodes the GCN4 domain

Nucleotides 262-1449 encodes the NA ectodomain

atgcccatggggtctctgcaaccgctggccaccttgtacctgctggggatgctggtcgct  M  P  M  G  S  L  Q  P  L  A  T  L  Y  L  L  G  M  L  V  Atccgtgctagcgtggagccacccgcagttcgagaaaggtggaggttccggaggtggatcg  S  V  L  A  W  S  H  P  Q  F  E  K  G  G  G  S  G  G  G  Sggaggtggatcgtggagccacccgcagttcgaaaaaagatctatgaaacaaatcgaagac  G  G  G  S  W  S  H  P  Q  F  E  K  R  S  M  K  Q  I  E  Daagctggaagaaatcctttcgaaactgtaccacatcgaaaacgagctggccaggatcaag  K  L  E  E  I  L  S  K  L  Y  H  I  E  N  E  L  A  R  I  Kaaactgctgggcgaaggatccgctgctggacagtccgtcgtgagcgtgaagctggccgga  K  L  L  G  E  G  S  A  A  G  Q  S  V  V  S  V  K  L  A  Gaacagcagcctgtgcccagtgagcggatgggccatctacagcaaggacaacagcgtgcgc  N  S  S  L  C  P  V  S  G  W  A  I  Y  S  K  D  N  S  V  Ratcggcagcaagggcgacgtgttcgtgatccgcgagcccttcatcagctgcagccccctg  I  G  S  K  G  D  V  F  V  I  R  E  P  F  I  S  C  S  P  Lgagtgccgcaccttcttcctgacccagggcgccctgctgaacgacaagcacagcaacggc  E  C  R  T  F  F  L  T  Q  G  A  L  L  N  D  K  H  S  N  Gaccattaaggaccgcagcccatacaggaccctgatgagctgccccattggagaggtgcca  T  I  K  D  R  S  P  Y  R  T  L  M  S  C  P  I  G  E  V  Pagcccatacaacagcaggtttgagagcgtggcttggtccgccagcgcttgccacgatgga  S  P  Y  N  S  R  F  E  S  V  A  W  S  A  S  A  C  H  D  Gatcaactggctgaccattggaatcagcggaccagacaacggcgccgtggccgtgctgaag  I  N  W  L  T  I  G  I  S  G  P  D  N  G  A  V  A  V  L  Ktacaacggcatcatcaccgacaccatcaagagctggcgcaacaacatcctgcgcacccag  Y  N  G  I  I  T  D  T  I  K  S  W  R  N  N  I  L  R  T  Qgagagcgagtgcgcctgcgtgaacggcagctgcttcaccgtgatgaccgacggccccagc  E  S  E C  A  C  V  N  G   S  C  F  T  V  M  T  D  G  P  Saacggccaggccagctacaagattttccgcatcgagaagggcaagatcgtgaagagcgtg  N  G  Q  A  S  Y  K  I  F  R  I  E  K  G  K  I  V  K  S  Vgagatgaacgcccccaactaccactacgaggagtgcagctgctaccccgacagcagcgag  E  M  N  A  P  N  Y  H  Y  E  E  C  S  C  Y  P  D  S  S  Eatcacctgcgtgtgccgcgacaactggcacggcagcaaccgcccctgggtcagcttcaac  I  T  C  V  C  R  D  N  W  H  G  S  N  R  P  W  V  S  F  NCagaacctggagtaccagatcggctacatctgctccggaatctttggagacaatcccagg  Q  N  L  E  Y  Q  I  G  Y  I  C  S  G  I  F  G  D  N  P  Rccaaatgacaagaccggcagctgcggaccagtgagcagcaatggagctaacggcgtgaag  P  N  D  K  T  G  S  C  G  P  V  S  S  N  G  A  N  G  V  Kggcttcagcttcaagtacggcaacggcgtgtggatcggccgcaccaagagcatcagcagc  G  F  S  F  K  Y  G  N  G  V  W  I  G  R  T  K  S  I  S  Scgcaacggcttcgagatgatctgggaccccaacggctggaccggcaccgacaacaacttc  R  N  G  F  E  M  I  W  D  P  N  G  W  T  G  T  D  N  N  Fagcatcaagcaggacatcgtgggcatcaacgagtggagcggatacagcggcagctttgtg  S  I  K  Q  D  I  V  G  I  N  E  W  S  G  Y  S  G  S  F  Vcagcacccagagctgaccggactggactgcatcaggccctgcttctgggtggagctgatc  Q  H  P  E  L  T  G  L  D  C  I  R  P  C  F  W  V  E  L  Iaggggaagacccaaggagaacaccatctggaccagcggcagcagcattagcttttgcgga  R  G  R  P  K  E  N  T  I  W  T  S  G  S  S  I  S  F  C  Ggtgaacagcgacaccgtgggatggagctggccagatggagctgagctgcccttcaccatc  V  N  S  D  T  V  G  W  S  W  P  D  G  A  E  L  P  F  T  I gacaagtga  D  K  -

Example 12 Expression of Foreign Proteins from the NSR Genome

Replicon cell lines expressing trimeric soluble hemagglutinin protein(sHA₃) and tetrameric soluble neuraminidase protein (sNA₄) frominfluenza virus H1N1 were produced essentially as described in Example 5with the modifications schematically depicted in FIG. 18. A repliconcell line expressing the eGFP protein was also produced alongside inthese experiments.

Flow cytometry analysis using an antibody specific for the N protein wasused to determine the percentage of cells containing both the L andS(S-eGFP, S-_(s)HA₃ or S-_(s)NA₄) genome segments. N protein expressionis dependent on the presence of both L and S genome segments. Flowcytometry demonstrated that 96% of the cells in which the L and S-eGFPgenome segments were introduced were positive for N protein expressionat passage 8 (FIG. 19A, results obtained from analysis of controlBHK-GnGc cells are depicted in FIGS. 19D, E and F). Flow cytometryanalysis of cells containing L and the S-CD5-HA-GCN4-ST (i.e. S-_(s)HA₃)segment demonstrated that 98% of the cells were positive for Nexpression (FIG. 19B). Flow cytometry analysis of cells containing L andthe S-CD5-OS-GCN4-NA segment (i.e. S-_(s)NA₄) demonstrated that 98.5% ofthe cells were positive for N expression (FIG. 19C).

Production of the sHA₃ and sNA₄ Proteins by the Replicon Cell Lines

Cell culture medium was collected and pre-cleared by slow-speedcentrifugation. Proteins were purified from the culture medium usingStrep-Tactin Sepharose gravity-flow columns according to theinstructions from the manufacturers (IBA, Göttingen, Germany). Theeluted fractions were analyzed by Silver-stained polyacrylamide gels(FIG. 20A) and Western blot using Strep-Tactin conjugated to horseradishperoxidase (Strep-Tactin-HRP, IBA, FIG. 20B).

The yield of both proteins was estimated at 1 mg/l of culture medium(BCA assay, Pierce, Thermo Scientific, Landsmeer, The Netherlands). Theproduction yields of the sHA₃ and sNA₄ proteins were again determinedafter cell passage 20 and found unchanged.

Transfection of the replicon cell lines with the pCAGGS-NSmGnGc plasmidresulted in NSR titers of 10⁷³ TCID50/ml of NSR-_(s)HA₃ and 10⁶TCID50/ml of NSR-_(s)NA₄. These particles will be used as vaccines forthe control of influenza.

The invention claimed is:
 1. A method for generating a recombinant,non-spreading bunyavirus replicon particle, the method comprising: A)culturing a eukaryotic cell with growth medium; B1) expressing T7polymerase in the eukaryotic cell; B2) expressing bunyavirus Gn proteinand bunyavirus Gc proteins, and optionally bunyavirus NSm protein, inthe eukaryotic cell, wherein said Gn and Gc proteins may form aheterodimeric polyprotein (GnGc); and wherein upon the optionalexpression of NSm protein, a heterotrimeric polyprotein (NSmGnGc) mayform; B3) transforming or transducing the eukaryotic cell with a vectorcomprising cDNA that encodes a bunyavirus L genome segment, wherein saidL genome segment cDNA is flanked by a T7 promoter and cDNA of a ribozymesequence; B4) transforming or transducing the eukaryotic cell with avector comprising cDNA that encodes at least part of a bunyavirus Sgenome segment, wherein said S genome segment cDNA comprises at leastthe N-gene and the 3′ and 5′ UTRs, wherein said S genome segment cDNA isflanked by a T7 promoter and cDNA of a ribozyme sequence; and,optionally, B5) transforming or transducing the eukaryotic cell with avector comprising cDNA that encodes a bunyavirus M genome segment fromwhich the GnGc coding region has been functionally inactivated, whereinsaid M genome segment cDNA is flanked by a T7 promoter and cDNA of aribozyme sequence; C) following steps B1-B5, the eukaryotic cell ismaintained in the growth medium and recombinant, non-spreadingbunyavirus replicon particles are generated and isolated from the growthmedium; wherein the sequence of steps B1, B2, B3, B4, B5 is random andall or part of these steps may be performed simultaneously.
 2. Themethod according to claim 1, wherein the T7 polymerase is provided tothe eukaryotic cell by transfected the eukaryotic cell with arecombinant fowlpox virus expressing said T7 polymerase.
 3. The methodaccording to claim 1, wherein the eukaryotic cell is provided with thebunyavirus proteins of step B2) through transfection of the eukaryoticcell with a recombinant non-replicative and/or non-spreadingparamyxovirus expressing said bunyavirus Gn, Gc, and/or NSm proteins. 4.The method according to claim 3, wherein the eukaryotic cell is stablytransfected or wherein the recombinant non-replicative and/ornon-spreading paramyxovirus is persistently present.
 5. The methodaccording to claim 1, wherein the eukaryotic cell is provided with thebunyavirus proteins of step B2) through transfection of the eukaryoticcell with an expression vector conditionally expressing said bunyavirusGn, Gc, and optionally NSm proteins.
 6. The method according to claim 1,wherein the bunyavirus L genome segment and/or the S genome segment,and/or, when present, the M genome segment comprises a foreign gene. 7.The method according to claim 1, further comprising a M, L, or Sminigenome, wherein a foreign gene is present in said M, L orS-minigenome.
 8. The method according to claim 1, wherein the eukaryoticcell is a mammalian cell.
 9. The method according to claim 1, whereinthe bunyavirus is Rift Valley fever virus.
 10. A recombinant,non-spreading bunyavirus replicon particle produced by the methodaccording to claim
 1. 11. The recombinant, non-spreading bunyavirusreplicon particle according to claim 10, wherein the bunyavirus L genomesegment and or the S genome segment and/or, when present, the M genomesegment, comprises a foreign gene.
 12. The recombinant, non-spreadingbunyavirus replicon particle according claim 10, wherein the bunyavirusis Rift Valley fever virus.
 13. A method for producing a bunyavirusreplicon particle, the method comprising: A) culturing a eukaryotic cellwith growth medium; B) transfecting, transforming, or transducing saideukaryotic cell with vectors expressing the bunyavirus NSm, Gn, and Gcproteins; C) transfecting the eukaryotic cell with the recombinant,non-spreading bunyavirus replicon particle according to claim
 10. 14. Amedicament or an immunogenic composition comprising the recombinant,non-spreading bunyavirus replicon particle according to claim
 10. 15.method according to claim 8, wherein the mammalian cell is BHK-21. 16.The method according to claim 8, wherein the mammalian cell is BSR-T7/5.